Nonpeptidic Amphiphilic Xanthone Derivatives: StructureâActivity

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Non-Peptidic Amphiphilic Xanthone Derivatives: StructureActivity Relationship and Membrane-Targeting Properties Jun-Jie Koh, Hanxun Zou, Shuimu Lin, Huifen Lin, Rui Ting Soh, Fang Hui Lim, Wee Luan Koh, Jianguo Li, Rajamani Lakshminarayanan, Chandra S. Verma, Donald T. H. Tan, Derong Cao, Roger W. Beuerman, and Shouping Liu J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01500 • Publication Date (Web): 03 Dec 2015 Downloaded from http://pubs.acs.org on December 8, 2015

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Journal of Medicinal Chemistry

Non-Peptidic Amphiphilic Xanthone Derivatives: Structure-Activity Relationship and MembraneTargeting Properties Jun-Jie Koh‡,†,¤, Hanxun Zou‡,†,§, Shuimu Lin†,§, Huifen Lin†, Rui Ting Soh†,∑, Fang Hui Lim†, Wee Luan Koh†, Jianguo Li†,¥, Rajamani Lakshminarayanan,†,# Chandra Verma†,¥,€,||, Donald T. H. Tan,¤,∑, Derong Cao,*, § Roger W. Beuerman*,†,# Shouping Liu*,†,# †

Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower Level

6, 169856, Singapore ¤

Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of

Singapore, 119074, Singapore ∑

#

Singapore National Eye Centre, 11 Third Hospital Avenue, 168751, Singapore

SRP Neuroscience and Behavioural Disorders, Duke-NUS Graduate Medical School,

169857, Singapore §

School of Chemistry and Chemical Engineering, South China University of Technology,

Guangzhou 510641, China ¥

Bioinformatics Institute (A*STAR), 30 Biopolis Street, 07-01 matrix, 138671, Singapore

€

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive,

637551, Singapore

1 ACS Paragon Plus Environment


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||

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4,

117543, Singapore KEYWORDS: Xanthone, structure-activity relationship (SAR), amphiphiles, antimicrobial, membrane targeting ABSTRACT

We recently reported the bio-inspired synthesis of a highly potent non-peptidic xanthone, 2c (AM-0016) with potent antibacterial activity against MRSA. Herein, we report a thorough structure-activity relationship (SAR) analysis of a series of non-peptidic amphiphilic xanthone derivatives in an attempt to identify more potent compounds with lower hemolytic activity and greater membrane selectivity. Forty-six amphiphilic xanthone derivatives were analyzed in this study and structurally classified into four groups based on spacer length, cationic moieties, lipophilic chains and tri-arm functionalization. We evaluated and explored the effects of the structures on their membrane-targeting properties. The SAR analysis successfully identified 3a with potent MICs (1.56 – 3.125 µ/mL) and lower hemolytic activity (80.2 µg/mL for 3a versus 19.7 µg/mL for 2c). 3a displayed membrane selectivity of 25.7 – 50.4. Thus, 3a with improved HC50 value and promising selectivity could be used as a lead compound for further structural optimization for the treatment of MRSA infection.

Introduction Antibiotic resistance is widely associated with the failure of clinical treatment, additional mortality and healthcare costs.1 In recent decades, bacterial resistance to commercial antibiotics has prompted interest in searching for and screening a new generation of antibacterial agents.2 Natural products are major sources of diverse bioactive compounds and have been crucial for the discovery and development of anti-infective agents.3 For instance, 2 ACS Paragon Plus Environment

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doripenem, tigecycline, retapamulin, telavancin and monobactam aztreonam are natural products or natural product-derived antimicrobials that have been approved by the FDA since 2005.3 Unfortunately, the development of bacterial resistance against these agents is inevitable.4 Therefore, there is still an urgent need for a sustainable pipeline to supply new and effective antibiotics without cross-resistance to currently used antibiotics.5 To overcome bacterial resistance, antimicrobials with new mechanisms of action are urgently needed.6 Traditional drugs such as β-lactams, aminoglycosides, tetracyclines, rifamycins, macrolides, glycopeptides, oxazolidinones and quinolones inhibit bacterial growth by interfering with an intracellular component of a biochemical pathway.6 However, these targets appear to be susceptible to the development of bacterial resistance due to the overuse and misuse of antibiotics. Bacteria develop resistance via several pathways, including bypassing the inhibited step, modifying the site of action, efflux mechanisms, target mutation and modification of cell wall permeability.7 Disrupting the bacterial inner membrane is lethal because the lipid membrane hosts many proteins that are essential to membrane functions, including energy metabolism and membrane potential.8 Membrane-targeting antimicrobials directly disrupt inner membrane integrity and may have reduced potential for the emergence of bacterial resistance,7 as suggested by multipassage resistance selection studies.8 Many generations of steady selection pressure are required to reconfigure the membrane to develop resistance against membranetargeting antimicrobials,9 making the development of resistance unlikely. Xanthone derivatives have been widely recognized as candidates for drug development. For instance, α-mangostin is identified as a compound with potent pharmacological activities,10 including antibacterial,8a anti-tumoral,11 anti-inflammatory12, antifungal,13 antioxidant14 and anti-allergy15 properties. α-Mangostin, an abundant xanthone extracted from Garcinia mangostana, exhibits potent antimicrobial activity against Gram-positive bacteria, including



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methicillin-resistant Staphylococcus aureus (MRSA), via a membrane-targeting pathway.8a However, the highly hydrophobic nature of the xanthone scaffold provides a strong driving force to penetrate both bacterial and mammalian membranes, resulting in low membrane selectivity.8a Cationic antimicrobial peptides (CAMPs) are critical components of the mammalian innate immune system that provide protection against pathogen invasion.16 CAMPs selectively inhibit and kill bacteria without inducing significant host cell cytotoxicity.17 Many CAMPs are also very specific to bacterial membranes over mammalian membranes.16a Interestingly, CAMPs have remained an effective agent against bacterial infection over evolutionary time.18 However, CAMPs have not found clinical use because they are costly to produce, salt sensitive, and unstable, with unresolved issues of systemic toxicity.19 Inspired by the amphiphilic topology of CAMPs, we recently reported a novel series of amphiphilic nonpeptidic xanthone derivatives with improved antimicrobial properties and membrane selectivity.20 Our preliminary studies indicated that cationic moieties with high pKa values are essential for disruption of the bacterial membrane by the xanthone derivatives at the hydrophobic-water interface.20 This design strategy was based on a pharmacophore model for short CAMPs with an amphiphilic topology in which hydrophilic and hydrophobic regions are segregated into opposing regions.21 Using this strategy, the membrane selectivity of the xanthone derivatives was successfully enhanced.20, 22 In the present study, we conducted follow-up structure-activity relationship (SAR) analyses with the aim of identifying novel potent amphiphilic xanthone derivatives with high or comparable membrane selectivity and reduced hemolytic activity compared with 2c (AM0016), using α-mangostin as a template. In this study, we report the design, antimicrobial activity and selectivity of a collection of forty-six non-peptidic amphiphilic xanthone derivatives, which were classified into 4 main categories based on differences in structure:


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spacer length, cationic end groups, varied lipophilic chains and tri-arm functionalization. We further report an extensive analysis and evaluation of their interactions with Gram-positive bacterial membranes using biophysical approaches. This SAR analysis enabled us to draw pertinent conclusions with respect to the structural parameters and physicochemical properties required for potency or high selectivity of amphiphilic xanthone derivatives. Chemistry Compounds 2a to 2h were designed to investigate the effects of the spacer length of amphiphilic xanthone analogs on their membrane-targeting properties. The syntheses of 2b, 2c, 2d and 2e were reported previously.20 As shown in Scheme 1, a similar strategy can be used to prepare compounds 2a, 2f, 2g and 2h using different α,ω-dibromoalkanes via Williamson ether synthesis, followed by amination with diethylamine. The preparation of 3a was accomplished by Gabriel synthesis. As shown in Scheme 2, the intermediate 1c was coupled with potassium phthalimide in anhydrous DMF (N,Ndimethylformamide) to afford N-alkylphthalimide 3 in good yield. Then, 3 was hydrolyzed using methylamine solution (33% in absolute ethanol) to produce the primary amine 3a. Next, 3b was synthesized from 3a and 1H-pyrazole-1-carboxamidine in a one-step reaction using a previously reported method.23 Purification of 3a and 3b by normal silica gel column chromatography was very difficult, and therefore these compounds were purified by preparative reversed phase HPLC. We further synthesized the tertiary amine compounds 4a4n and 5a-5k and aromatic amine compounds 6a-6c to study the effect of the cationic end group on antimicrobial activity (Scheme 3). Compounds 5a, 5c and 5e were synthesized according to the previous method.20 All other compounds were synthesized using a similar protocol. Briefly, intermediate 1c was coupled with diverse aliphatic secondary amines in DMSO (dimethyl sulfoxide) for 4 h to obtain 4a-4n and 5a-5k. All amination reactions were



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performed at room temperature, except 4b. To synthesize 4b, the reaction mixture was heated to 50 °C because diethanolamine is less reactive than the other secondary amines. Compounds 6a to 6c were synthesized according to a previously reported method22 using 1c and imidazole, pyrazole or 1,2,4-triazole, respectively. The reactions were conducted in the presence of K2CO3 in acetone under reflux conditions for 48 h. In Scheme 4, the xanthone analogs 8a and 8b without isoprenyl groups at the C3 and C6 positions were synthesized via condensation of 2,4-dihydroxylbenzoic acid and phloroglucinol in the presence of Eaton’s reagent. Xanthone 7 was reacted with 1,4dibromoalkane to afford xanthone intermediate 7a, followed by amination reaction with secondary amines to afford compounds 8a and 8b. The syntheses of the tetrahydro α-mangostin derivatives 10a to 10c are outlined in Scheme 5. The double bonds of the isoprenyl groups were reduced by catalytic hydrogenation to afford tetrahydro α-mangostin 9 in good yield. Then, 9 was alkylated with 1,4-dibromobutane to produce intermediate 9a. The treatment of 9a with three different secondary amines in DMSO produced compounds 10a, 10b and 10c in good yields. The hydroxyl group at the C1 position in the α-mangostin scaffold is less reactive due to the formation of an intramolecular hydrogen bond between the C1 hydroxyl group and the C9 carbonyl group20. Thus, harsher conditions are required for C1 amination. To synthesize triarm functionalized α-mangostin derivatives, we used the more reactive electrophilic reagent 1,4-diiodobutane instead of 1,4-dibromobutane to form intermediate 11. Then, 11 was aminated using diethylamine in DMSO to obtain tri-arm functionalized 12. These reactions are described in the Experimental Section. Results and Discussion Design of amphiphilic xanthone derivatives. We previously coupled cationic moieties with different pKa values to the C3 and C6 positions of the xanthone scaffold to obtain an 6 ACS Paragon Plus Environment

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amphiphilic structure.20 A key feature in the design was that the cationic moieties had high pKa values of ≥ 10.5, with a clear segregation of cationic and hydrophobic regions. 2c displayed the most potent antimicrobial activity (MIC = 0.39 – 1.56 µg/mL) and membrane selectivity (HC50/MIC; mostly in the range of 13 – 50). Therefore, in this study, 2c was selected as the lead compound for further structural modification. In general, 2c can be conceptually divided into 4 distinct portions: spacer length, cationic end groups, varied lipophilic chains and tri-arm functionalization. The structure-activity relationship that emerged from these portions in this study is summarized in Figure 1. SAR: Effect of spacer length We previously reported that diethylamine-modified xanthone derivatives with spacer lengths of 3 – 6 carbons displayed MIC values of 0.39 – 1.56 µg/mL. Based on these results, we first investigated the effect of spacer length by preparing new compounds with different spacer lengths of 2, 8, 10 and 12. The MIC values of these xanthone analogs against four different strains of Gram-positive bacteria are presented in Table 1 (clinical isolates S. aureus DM4001, MRSA DM9808R, MRSA DM21455 and B. cereus ATCC11778). Compounds 2b – 2e (n = 3 – 6) displayed potent antimicrobial activities against all strains tested. Surprisingly, for all compounds with spacer lengths of n = 2 and n ≥ 8, no inhibition was observed against all strains tested at the highest tested concentration of 50 µg/mL (Table 2). Next, we investigated the cytoplasmic membrane-targeting properties of the compounds in live bacterial cells. SYTOX Green is a membrane impermeant, DNA-binding dye that stains bacterial cells with damaged membranes, as indicated by an increase in fluorescence. In addition, the influx of SYTOX Green is a good indicator of large scale-membrane disruption due to its larger size.16b Figure 2 shows that S. aureus DM4001 treated with 2b – 2e (n = 3, 4, 5 and 6) at 10 µM showed significant SYTOX Green influx in 200 s. Compounds 2a (n = 2) and 2f (n = 8) also promoted dye uptake but at a much slower rate. By contrast, 2g (n = 10) 7 ACS Paragon Plus Environment


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and 2h (n = 12) caused only very small amounts of dye influx. These data correlate well with the MIC values and demonstrate that membrane permeabilization by 2b – 2e is crucial for the induction of bacterial cell death. To further evaluate the effects of the spacer length of the xanthone derivatives on membrane integrity, the Live/Dead BacLight bacterial viability kit was used. The BacLight reagent uses two nucleic acid stains with different properties to distinguish cells with damaged cell membranes from cells with intact cell membranes. SYTO-9 is a greenfluorescent nucleic acid that stains bacteria with either intact or damaged membranes. By contrast, propidium iodide is a red-fluorescent nucleic stain that penetrates only bacteria with damaged membranes and reduces the fluorescence intensity of SYTO-9. As shown in Table 2, diethylamine-modified xanthone derivatives (2b – 2e) with spacer lengths of n = 3 – 6 significantly reduced the integrity of the bacterial membrane to < 50%. 2c (n = 4) was the most effective compound and reduced membrane integrity to 37.8 ± 5.1%. 2a (n = 2) was less efficient in reducing membrane integrity (64.1 ± 1.2%). Nearly no reduction in membrane integrity (> 95.5%) was detected for bacteria exposed to 2f – 2h (n = 8, 10 and 12). These results suggest that active amphiphilic xanthone analogs with spacer lengths of 3 – 6 (2b – 2e) killed the bacteria via inner membrane disruption.24 The potent activities of compounds with spacer lengths of n = 3 to 6 were clearly defined. Spacer lengths that were too short (n = 2) or too long (n ≥ 8) were detrimental to antibacterial activity for reasons that are unclear. Because 2a – 2h were all modified with diethylamine with the same pKa value, the results suggest that the spacer length may play an important role in their antibacterial action. For 2a (n = 2), we postulate that the distance between the oxygen atoms at the C3 and C6 positions and the terminal nitrogen atoms are sufficiently short to permit a significant inductive effect of the oxygen atoms. The effect of the spacer length on electron density was studied by 1H NMR spectra, as shown in Figure 3. For compounds with


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spacer lengths of n = 5 and longer, the Hb proton signals were observed at 2.40 – 2.48 ppm, whereas the Ha proton signals were downfield at 2.50 – 2.60 ppm. At spacer lengths of n = 4 (2c), the signals of the Hb protons were shifted downfield and merged with the signals of the Ha protons at 2.50 – 2.55 ppm. The Hb proton signal was further shifted downfield when the spacer length was reduced from n = 4 to n = 2. For 2a, which has a spacer length of n = 2, the Hb proton signals were significantly shifted downfield (3.00 ppm). By contrast, the Ha proton signal was not significantly shifted. The results clearly demonstrate that the strong inductive effect of the oxygen atom has a significant effect on the electron distribution of the Hb proton attached to the carbon adjacent to the amine group when the spacer length is two in 2a. The pKa value of the diethylamine in 2a (n = 2) is expected to be smaller than its normal value of 10.98 in 2c (n = 4), and thus the antimicrobial properties of 2a are much weaker than those of 2b – 2e (Table 2). In support of this interpretation, Table 3 presents the effects of the spacer length between the amine and alcohol groups on the pKa value. The pKa values of alkanolamines with n = 3 (1,3-propanolamine, dimethylpropanol amine and diethylpropanolamine, with pKa values of 9.91, 9.27 and 10.14, respectively) are higher than those of the respective alkanolamines with n = 2 (monoethanolamine, dimethylethanolamine and diethylethanolamine, with pKa values of 9.16, 8.88 and 9.79, respectively).25 The pKa shifts are -0.76, -0.39 and -0.35, respectively. Therefore, the pKa of diethylamine of 2a was expected to be lower. Cationic charge is important for the distribution of membrane-targeting antimicrobials on the bacterial surface and subsequent disruption of the bacterial membrane.16a Consequently, 2a has a weaker ability to disrupt the inner membrane, as shown by the SYTOX Green uptake (Figure 2) and BacLight studies (Table 2). The inductive effect and pKa profile could not explain the reduced antimicrobial properties of the compounds with longer spacer length (n ≥ 8), however, because no significant proton



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shifts were observed in their NMR spectra (Figure 3). One possible explanation for this observation is molecular aggregation. A number of studies report that longer alkyl chains facilitate aggregation.26 For instance, Malina and Shai have reported that the enhanced oligomerization of a compound can endow it with a large volume, limiting its access to penetrate to the cytoplasmic membrane via the bacterial cell wall, as in peptidoglycan.27 Aggregation reduces the effective concentration of xanthone derivatives with longer spacer lengths in solution, thus reducing hemolytic activity with increasing spacer length when n ≥ 8 (Table 2). The thermodynamics of aggregation of the xanthone derivatives was examined using atomistic MD simulations. In this study, five xanthone derivatives with different spacer lengths were chosen: n = 2, 3, 6, 8 and 12. Because any chemical process is governed by the change in Gibbs free energy, the ∆G of aggregation for each of the five xanthone derivatives was calculated using metadynamics MD simulations. Figure 4 shows ∆G as a function of the distance between the two monomers. All five xanthone derivatives displayed aggregation tendencies, as revealed by the free energy well at approximately 0.42 nm, which was driven by the aromatic stacking of the central hydrophobic core. Moreover, the aggregation tendency was strongly correlated to the spacer length. For instance, in 2h (n= 12), the long aliphatic chain significantly contributed to aggregation at longer distances, as seen from the broader free energy well in the potential of mean force. These results suggest that 2h can form much larger and tighter aggregates than other derivatives with shorter spacer lengths. The aggregation of antimicrobials in solution is widely associated with reduced antimicrobial performance. Aggregation reduces the interaction of the antimicrobial with its target, thereby reducing antibacterial potency.28 In addition, large supramolecular aggregates in solution are also prohibited from reaching the cytoplasmic membrane because they cannot pass through the bacterial outer membrane.29 To examine the effect of xanthone aggregates 10 ACS Paragon Plus Environment

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on the inner membrane, the ability of these compounds to disrupt calcein-loaded liposomes (DOPE/DOPG= 75/25), which mimic the bacterial membrane, was studied. The liposome is a good mimic of the lipid membrane but not a real bacterial model because the bacterial membrane also consists of an outer membrane. In contrast to the SYTOX Green uptake and BacLight assays, which utilize whole cells, the calcein leakage study (Figure 5) revealed that the lytic activities of 2f (n = 8) and 2g (n = 10) were similar to that of 2c (n = 4), the most potent compound in this series in inducing calcein leakage from LUV. At compound to lipid ratios of 0.125 – 0.5, 2c, 2f and 2g all induced > 70% leakage. 2h (n = 12) exhibited slightly weaker lytic activity, with approximately 60% leakage. These results suggest that xanthone molecules in their aggregated state can disrupt lipid mimics of the inner membrane but cannot reach the cytoplasmic membrane because their aggregates cannot penetrate the bacterial outer membrane. Consequently, aggregation leads to low partitioning into the membrane, consistent with the experimental observation that xanthone derivatives with longer spacer lengths exhibit reduced membrane perturbing activity for both bacterial and mammalian membranes. These results are also consistent with a report from Sarig et al. that tighter and more stable aggregates are responsible for reduced potency in aqueous media.28 Thus, the SYTOX Green uptake and calcein leakage assays also permit the distinction between truly inactive xanthone analogs and potentially active ones. The hemolytic activity of compounds 2f and 2g further supports this finding. Combining these biophysical studies revealed two important issues. First, amine moieties with high pKa values are crucial for interaction with the negatively charged bacterial membrane via electrostatic interaction, as suggested by the surface activity model.30 Electrostatic interactions are crucial to attract the cationic xanthone derivatives to the membrane surface, regardless of their spacer length. A high surface concentration of xanthones in the outer leaflet will result in membrane disruption. Second, to perturb the inner 11 ACS Paragon Plus Environment


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membrane, amphiphilic xanthone derivatives must be able to penetrate the peptidoglycan layer of Gram-positive bacteria effectively. Xanthones with longer spacer lengths tend to aggregate and have more difficulty passing through the peptidoglycan layer. Hence, although strong leakage in liposomes was observed for compounds with spacer lengths ≥ 8, they did not exhibit antibacterial activity. SAR: Effect of cationic moieties. Next, modifications of the cationic R group moieties were explored. Based on our previous study, we first examined the effect of introducing a primary amine group in 3a to replace N,N-diethylamine (2c). As shown in Table 4, this modification resulted in a significant improvement in hemolytic activity (HC50 = 80.2 µg/mL for 3a versus 19.6 µg/mL for 2c) while maintaining good in vitro antibacterial activity (MIC = 1.56 µg/mL). The selectivity of 3a (51.4) was also very similar to 2c (50.4). Replacements using guanidine (3b), N,Ndimethylamine (4a) and N,N-methylethylamine (4c) did not seem to provide any benefit because these compounds exhibited 4- and 8-fold lower potency as well as lower selectivity (10.3 – 20.5). We explored the effect of introducing two hydroxyl groups to N,Ndiethylamine (4b). The pKa of 4b was dramatically reduced to 8.88 due to the inductive effect of the two hydroxyl groups. As expected, its MIC was reduced 16-fold compared to 2c. In addition, no improvement in selectivity was observed. A similar effect was observed for 4e containing only one hydroxyl group because the MIC and selectivity were both reduced significantly to 6.25 µg/mL and 5.1, respectively. These results suggest that a hydroxyl group is detrimental to the antibacterial activity of amphiphilic xanthone analogs. Next, we replaced N,N-diethylamine with its isomer, N,N-methylpropylamine (4d). Surprisingly, this replacement negatively affected in vitro potency; a 64-fold reduction of antibacterial activity from 0.39 µg/mL for 2c to 25 µg/mL for 4d was observed. Because N,N-diethylamine and N,N-methylpropylamine have similar pKa values, this result suggests


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that the pKa of the cationic moieties is not the sole factor affecting the antibacterial activity of amphiphilic xanthones. The small steric hindrance of the tertiary amines in 2c may play a positive role in antimicrobial activity and selectivity compared with the greater steric hindrance of N,N-methylpropylamine in 4d. To investigate the effect of steric hindrance on the antibacterial activities of the amphiphilic xanthone analogs, further modification was focused on increasing the alkyl length of N,Ndialkylamine. In general, when N,N-diethylamine was replaced with N,N-dialkylamine with a total carbon number > 4, significantly reduced potency was observed. For instance, xanthone derivatives coupled to N,N-butylmethylamine (4f), N,N-ethylpropylamine (4g), N,Nethyllisopropylamine (4h), N,N-pentylmethylamine (4i), N,N-butylmethylamine (4j), N,Ndipropylamine (4k) and N,N-hexylmethylamine (4l) displayed poor MIC values of 12.5 – 50 µg/mL. The loss of activity (MIC > 50 µg/mL) observed for 4m and 4n clearly indicates that substituents with a total carbon number of 8 were deleterious. Careful evaluation of the potency of these compounds revealed that N-ethyl-substitution was the optimal alkyl length of the cationic moieties for potent MIC values (0.39 – 3.125 µg/mL). Any substitution with N-propyl or a longer alkylamine diminished the antibacterial activity (MIC ≥ 12.5 µg/mL). Next, we turned our attention to saturated heterocyclic cationic moieties (compounds 5a – 5k). Table 4 shows that the compounds, except 5a and 5c, were much less potent than 2c, with MIC values ≥ 6.25 µg/mL. These compounds also displayed weak selectivity; the maximum attainable selectivity achieved was < 25 for all compounds studied. In general, pyrrolidine- (5a) and piperidine- (5c) coupled xanthones displayed the most potent antimicrobial properties, with MIC values of 0.78 – 1.56 µg/mL. When a hydroxyl group was introduced to the 4-position of piperidine (5d), an 8-fold reduction of potency was observed, with an MIC value of 6.25 µg/mL. This result further supports our earlier observation that substitution with a hydroxyl group reduces the antimicrobial activity and membrane



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selectivity of amphiphilic xanthone derivatives. Replacing the piperidine with lower pKa morpholine (5e) and thiomorpholine (5f) led to a significant loss of activity (MIC > 50 µg/mL). A piperazine-coupled derivative (5g) had an MIC value of 6.25 µg/mL, an 8-fold reduction in potency compared to 5c (MIC: 0.78 µg/mL). A more hydrophobic derivative, 5h, with 1-methylpiperazine also displayed similar antibacterial activities (MIC = 6.25 µg/mL). Increasing the alkyl length to 1-ethylpiperazine (5i) further dramatically reduced activity to 25 µg/mL. Due to the low pKa values, other saturated heterocyclic-substituted derivatives, 5b, 5j, and 5k, also displayed less potent antibacterial activities, with MIC values of 12.5, 6.25 and > 50 µg/mL, respectively. Because we previously demonstrated that aromatic substitution is detrimental to the antibacterial activity of amphiphilic xanthone analogs,20 only limited examples of this group were explored in the present study. Table 4 shows that all derivatives in this group were inactive even at the highest concentration tested (> 50 µg/mL). In addition, no hemolytic activity was observed. In general, aromatic moieties have lower pKa values. For instance, 1H-imidazole, 1H-1,2,3-triazole and 1H-pyrazole have pKa values of 7.05, 2.20 and 2.52 respectively. Delocalization of electrons into the substituted aromatic rings greatly reduced the protonation of the aromatic amines, resulting in poorer interaction with the negatively charged bacterial membrane. The membrane-targeting properties of compounds 3a – 3b, 4a – 4n, 5a – 5j and 6b – 6c were tested against S. aureus DM4001 at 1× MIC. We first investigated the membrane permeabilization properties using the SYTOX Green uptake assay (Table 5 and Figure 6). The most striking permeabilization was observed for substituents with N,N-dialkylamine, with a total carbon number of 4 – 5 (4d – 4h; 80000 – 115000 c.p.s.). When the carbon number was further increased to 6 – 7 (compounds 4i – 4l), the extent of membrane permeabilization was reduced slightly to 45000 – 75000 c.p.s. When the carbon number


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reached 8 (4m and 4n), poor permeabilization with 1768-1778 c.p.s. was observed. We further investigated the leakage of ATP from bacteria using ATP determination kits in ATPfree PBS buffer. As shown in Table 5, strong ATP leakage was observed when S. aureus was incubated with xanthone derivatives coupled with N,N-dialkylamine with a carbon number of 4 – 7. Similar to the SYTOX Green uptake assay, only a low level of ATP leakage was detected when the bacteria were incubated with xanthone analogs modified using N,Ndialkylamine with a carbon number of 8. This observation is consistent with a previous report that extensive membrane perturbation is frequently followed by the leakage of larger cellular constituents such as ATP.31 Table 5 shows that, in general, hydrophobicity of compound increases with carbon number of the substituted moiety. In addition, moieties with the same carbon numbers have similar cLogP values. The exception was compound 4h, which have a similar cLogP value to 2c and 4d. In addition, Figure 7A shows that a significantly positive correlation was observed between MIC and the carbon number of N,N-dialkylamine for compounds 2c, 3a – 3b, 4a – 4n, with an rs value of 0.7948 (p = 0.0001). Carbon number emerged as an important parameter influencing MIC, suggesting that it is more difficult for compounds with high carbon numbers, e.g., 7 – 8, to approach the negatively charged bacterial membrane efficiently. Long alkyl substitution may sterically hinder the approach of the cationic nitrogen to the negatively charged membrane. For instance, as shown in Figure 6, 4f, 4i, 4k and 4l induced membrane permeabilization slowly. Therefore, a longer time was required to reach an adequate surface concentration in the outer leaflet of the membrane. Once an adequate concentration was achieved on the bacterial membrane, compounds with large carbon numbers of N,N-dialkylamine induced stronger membrane permeabilization or membrane disruption. For compounds coupled with N,N-dialkylamine with a carbon number > 8, the overall amphiphilic conformation of the xanthone derivatives was disrupted because the N,N-



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dialkylamine was too steric or hydrophobic. Therefore, no antibacterial activity was observed for 4m and 4n. Consistent with this result, Liu et al. reported that membrane destabilization through transmembrane pore formation requires an alkyl chain with sufficient length.32 In addition, Lin et al. also demonstrated that the alkylation of a membrane-perturbing peptide induces pores with larger size than those formed by a non-alkylated peptide.26a Figure 7B also indicates that there was a significant positive correlation between ATP leakage and carbon number for compounds 2c, 3a – 3b and 4a – 4l (Figure 7C, rs = 0.746, p = 0.0014). As shown in Table 6, heterocyclic-coupled xanthone compounds generally displayed weaker membrane permeabilization. These data suggest that cationic moieties with a saturated cyclic structure bulkier than N,N-dialkylamine may hinder membrane disruption by the amphiphilic xanthone. Overall, our findings suggest that the cationic moieties of the xanthone derivatives are important for selective interaction with bacterial cells and further perturb the negatively charged microbial cytoplasmic membrane. Amine moieties with high pKa values were required as they were most likely to be protonated to cationic charge. Therefore conjugation of these functional groups to the hydrophobic xanthone core would be more likely to result in compounds with more amphiphilic properties. In addition, amine moieties with high pKa values can increase the ratio of ionized amine to unionized amine at the pH of the physiological condition (pH= 7.4). These ionized molecules were readily to enter bacterial membrane to exert their membrane targeting mechanism. Then, the equilibrium would shift to produce more ionized molecule to target the bacterial membrane. These data also further support our earlier postulation that amphiphilic xanthone derivatives disrupt the bacterial membrane via an interfacial activity model, as described by Wimley.30 The carbon number of the high-pKa N,N-dialkylamine is also an important factor affecting antimicrobial activity. For heterocyclic-coupled xanthone derivatives, pKa is more crucial for good antimicrobial


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properties. Our SAR effort has identified 3a, which demonstrated comparable selectivity to 2c. In addition, 3a displayed a better hemolytic profile than 2c. SAR: Effect of varied lipophilic chains. As presented in Table 7, the removal of two isoprenyl groups (compounds 7, 8a and 8b; R1 and R2= H) resulted in a loss of antibacterial potency (MIC > 50 µg/mL) and a loss of hemolytic activity (HC50 > 200 µg/mL). These data strongly suggest that the removal of isoprenyl groups is not well tolerated by amphiphilic xanthone derivatives. We further modified the isoprenyl groups of compounds 1, 2c, 4a and 5a via hydrogenation to yield the corresponding compounds 9 and 10a – 10c, respectively. In general, 9 and 10a – 10c exhibited decreased potency; in particular, 9 exhibited a > 25-fold reduction of activity compared to 1 (> 50 µg/mL). In addition, these compounds also displayed slightly weaker hemolytic activity compared to their analogs without hydrogenation. In summary, antibacterial and hemolytic activities increased in the following manner: analogs without lipid substitutions (isoprenyl groups or hydrogenated isoprenyl) < analogs with hydrogenated isoprenyl groups < analogs with isoprenyl groups. We previously demonstrated that isoprenyl groups or lipophilic chains of amphiphilic xanthone analogs are crucial to enhance the interaction with the bacterial membrane.8a, 20 However, their membrane-targeting properties have yet to be investigated. We speculated that the relatively weaker membrane activities of these compounds were due to their poorer interaction with the inner membrane. To examine this possibility, we created calcein-loaded liposomes with different compositions, DOPE/DOPG = 75/25 and DOPC, to mimic the bacterial membrane and mammalian membrane, respectively. Then, the calcein leakage induced by 1, 2c and 4a, as well as their corresponding analogs with varied isoprenyl groups, was measured (Figure 8). Figure 8A and 8D show that 1, which has two isoprenyl groups, induced 29.4±3.0% and 33.6±11.0% leakage from liposomes with compositions of



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DOPE/DOPG = 75/25 and DOPC, respectively, at a lipid to compound (L/C) ratio of 4. The observed leakage decreased to 16% and 14.1±4.5% at an L/C ratio of 8. Further increasing the L/C ratio to 16 or higher resulted in leakage of < 10%. Notably, 7 (1 without isoprenyl groups) and 9 (1 with hydrogenated isoprenyl groups) only induced negligible calcein leakage from both liposomes at all L/C ratios tested, consistent with their reduced potency, as indicated by MIC > 50 µg/mL and HC50 > 200 µg/mL. Similar trends were observed for the lytic activities of 2c (Figure 8B and 8E). 2c induced significant leakage against both liposomes up to an L/C ratio of 16, at which leakage of > 70% was detected. 10a, the hydrogenated analog of 2c, exhibited reduced lytic potency relative to 2c. For instance, 10a only induced 78.0 ± 0.7%, 58.6 ± 4.3% and 20.1 ± 2.9% leakage against the bacteriamimicking membrane at L/C ratios of 4, 8 and 16. Consistent with the MIC and HC50 values, no detectable leakage was measured for 8a, a compound without isoprenyl groups, in both liposomes. Figure 8C shows that both 4a and 10b exhibited similar lytic activity against DOPE/DOPG = 75/25 liposomes, whereas 4a induced stronger lytic activity at L/C ratios of 16 – 64 compared to 10b against DOPC liposomes. Taken together, the lytic activities correlated well with the MIC and HC50 values of these analogs with varied isoprenyl groups. Therefore, our results further demonstrate that isoprenyl groups are crucial and enhance the interaction of amphiphilic xanthone analogs with the biological membrane. SAR: Effect of tri-arm functionalization. Finally, we explored the tri-arm functionalization of 2c by conjugating an extra N,Ndiethylamine at the C1 position. The hydroxyl group at the C1 position is relatively inert because of the potential for intramolecular hydrogen bond formation between the C1 hydroxyl group and the C9 carbonyl group. Therefore, more drastic reaction conditions were needed to obtain tri-arm 12 from 2c via 11 (Scheme 6).


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Table 8 shows that 12 displayed an MIC value of 12.5 µg/mL against S. aureus, which was 32-fold higher than the MIC value of 2c (0.39 µg/mL). The HC50 value of 12 was > 200 µg/mL. It is noteworthy that 12 is the only non-hemolytic xanthone analog (HC50 > 200 µg/mL) that displayed a reasonable MIC value (12.5 µg/mL). By contrast, nearly all other non-hemolytic xanthone analogs (4i, 4k – 4n, 5e, 5f, 5k, 6a – 6c) were very weak or inactive against. S. aureus (MIC ≥ 25 µg/mL). Next, the membrane-targeting properties of 12 were investigated. Figure 9 shows that 12 induced much weaker membrane permeabilization (SYTOX Green uptake = 4305.1 ± 46 c.p.s.) than 2c. The results suggest that the intramolecular hydrogen bond between the C1 hydroxyl group and the C9 carbonyl group was important for the formation of a “pseudo-ring” that is rigid and co-planar with the 3-membered ring xanthone and enhances membrane disruption. This 4-membered ring structure is similar to the 4-membered sterol core of the cationic steroidal antimicrobial CSA-13 (13) (ceragenin),33 which also displays potent antimicrobial activity via membrane targeting (see Figure 9 for the structure of 13). Taken together, these results indicate that the 4-membered ring structure of amphiphilic xanthones is crucial for membrane disruption. However, the results support the view that tri-arm modification at the C1, C3 and C6 positions may result in a potent xanthone analog with much lower hemolytic properties. In addition, the current biophysical study did not fully elucidate the membrane-targeting properties of 12. Further extensive SAR studies and biophysical studies investigating tri-arm-modified xanthone analogs are needed to clarify these issues. Summary of the structure-membrane targeting relationship In general, the bacterial membrane has two layers: the outer membrane and the inner membrane. Peptidoglycan is a major constituent of the outer membrane of Gram-positive bacteria. The bacterial inner membrane contains approximately one-third of the proteins in



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the cell and is critical for cellular processes, regardless of the metabolic status of the cell. The three major phospholipid species in the phospholipid bilayer are phosphatidylethanolamine (PE, 75%), phosphatidylglycerol (PG, 20%) and cardiolipin (diphosphatidylglycerol, CL, 1 – 5%). PG and CL are negatively charged, whereas PE is zwitterionic and neutral in charge. Gram-positive bacteria generally have a high content of the anionic lipids PG and CL.34 This study establishes the excellent structure-membrane targeting relationships of the amphiphilic xanthone analogs. Four distinct structural parameters were identified for membrane-targeting properties: spacer length, cationic moieties, lipophilic chains and tri-arm functionalization at the C1 position (Figure 1). The SAR study suggested that spacer lengths of 3 – 6 are optimal to avoid the inductive effect of the oxygen atom and to efficiently penetrate across the outer membrane. The inductive effect and aggregation are the major factors limiting the membrane-targeting properties of amphiphilic xanthone derivatives with short (n = 2) and long spacer lengths (n ≥ 8), respectively. The outer membrane is the major barrier excluding amphiphilic xanthone analogs with spacer lengths of n ≥ 8. The cationic moieties (R group) of the amphiphilic xanthone analogs are important for the interaction with the negatively charged bacterial inner membrane on the outer leaflet via electrostatic interaction. The optimal carbon number of the R group was ≤ 4 to avoid steric hindrance and exert strong membrane targeting action, and N,N-diethylamine and primary amine are the optimal cationic moieties. Isoprenyl groups at the C2 and C8 positions are important to exert a driving force to bring the amphiphilic xanthone derivatives into the inner leaflet of the bacterial membrane.35 The removal or hydrogenation of isoprenyl groups results in poorer antibacterial activities and reduced membrane targeting. Tri-arm functionalization at the C1 position also reduces the membranetargeting ability of the amphiphilic xanthone derivatives. In vitro antimicrobial potency and in vitro cytotoxicity of 3a


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Our extensive SAR analysis has identified 3a as a potential lead compound. 3a was further examined as small-molecule membrane-active antibiotics for its antimicrobial potency using time-kill kinetic assay. In this study, vancomycin was used as a comparator. Figure 10 shows that 3a killed bacteria quickly. For instance, 3a achieved a 3-log reduction of viable cell count (99.9% of bacteria killed) in 30 and 300 min respectively at 4× MIC and 2× MIC, respectively against S. aureus DM4001. Similar observation was observed for MRSA DM21455. At both 2× and 4× MIC, a 3-log reduction could be achieved in 30 min. By contrast, vancomycin induced negligible bacterial killed up to 300 min. Toxicity toward mammalian cells is a concern for some membrane-targeting antimicrobials with low membrane selectivity. In addition, a compound with low cytotoxicity is important for further clinical development. To evaluate cytotoxicity of 3a at its antimicrobial concentrations, luminescent cell viability (ATP assay) and cytotoxicity assay (LDH assay) with human corneal fibroblasts were used (Figure 11). The results revealed that 3a induce negligible cytotoxicity up to 4× MIC. In our recent report,22 we have shown that 2c is not active in an animal model of corneal infection. The main reason is that the tolerated dose of 2c is very low. Only 0.02% 2c could be applied and a less than 1 log reduction at the tolerated concentration could be achieved. By contrast, amino-acid (e.g., arginine) modified xanthone derivatives have higher tolerated dose, thus higher concentrations could be used in animal study. However, synthesis of aminoacid modified xanthone derivatives are more complicated. In this study, an intensive study on non-peptidic xanthone derivatives was performed. 3a with much improved HC50 than 2c was identified, which a higher tolerated does could be expected. 3a is also easier to be synthesized than amino-acid modified xanthone derivatives. The promising antimicrobial activity, improved selectivity, fast time-kill and low toxicity in vitro at antimicrobial concentration of



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3a suggest that this molecule is a new amphiphilic xanthone analog to combat serious forms of Gram-positive bacteria, including MRSA. Conclusion In this study, we synthesized a series of novel non-peptidic xanthone derivatives and evaluated their membrane-targeting properties. Our extensive SAR analysis further identified 3a, a potent xanthone analog of 2c with acceptable selectivity. 3a displays lower hemolytic activity than 2c and will be further evaluated for its in vivo toxicity and in an infection model. 12 was also identified as an active but non-hemolytic amphiphilic xanthone analog. Thus, 3a and 12 could be used as a lead compound for further structural optimization for the treatment of MRSA infection. Taken together, these results provide further valuable insight into the pharmacophores of xanthone compounds for targeting the bacterial cytoplasmic membrane. The SAR analysis presented here providing important strategy for a sustainable supply of new, effective, and safe antimicrobials without cross-resistance to currently used antibiotics. Particularly, many compounds designed in this study were active against MRSA strains. These results may lead to a new and effective class of amphiphilic xanthone analog to combat emerging antibiotic resistance. Experimental Section General Chemistry. α-Mangostin (purity 99.4%) was purchased from Chengdu Biopurify Phytochemicals Ltd, Chengdu, China. All starting materials and solvents were purchased from Sigma Aldrich unless otherwise stated and were used without further purification. Merck TLC aluminum-backed sheets (silica gel 60 F254) were used for TLC. Column chromatography was conducted using Merck 230−400 mesh silica gel 60. Chromatographic separation was achieved using preparative reverse phase HPLC on a Shimadzu LC-20AP with a C18 column (Phenomenex, 150×21.2 mm, 5 µ, 100 Å), with a flow rate of 10 mL/min, and UV detection at 254 nm. A mixture of water and methanol (both containing 0.1% formic


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acid) was used as the eluent. 1H NMR and

13

C NMR spectra were recorded on a Bruker

Avance 400 MHz with TMS as an internal standard. 1H NMR spectra are reported in parts per million (ppm) relative to the peak of CDCl3 (7.26 ppm), MeOD (3.31 ppm) or DMSO-d6 (2.50 ppm); 13C NMR spectra are reported in parts per million (ppm) relative to the central peak of CDCl3 (77.16 ppm), MeOD (49.00 ppm) or DMSO-d6 (39.50 ppm). Coupling constants (J) are reported in hertz (Hz), and peak multiplicities are reported as singlet (s), doublet (d), triplet (t), quadruplet (q), broad (br), and multiplet (m) where applicable. APCI mass spectra were measured using a Bruker Amazon; ESI mass spectra were measured using an API2000 LC/MS/MS system. The purity of the compounds was checked by analytical reverse phase HPLC on a Shimadzu LC-20AP with a C18 column (Phenomenex, 150×4.6 mm, 3 µ, 110 Å), with a flow rate of 0.7 mL/min, and UV detection at 254 nm. A mixture of water and methanol (both containing 0.1% formic acid) was used as the eluent. All compounds possessed purity above 95%. Melting points were recorded on a SRS Optimelt Automated Melting Point System, SRS MPA100 and are reported as uncorrected. General Procedure A for the Synthesis of Compounds 2a – 2g Potassium carbonate (5.0 equiv) and α,ω-dibromoalkane (15.0 equiv) were added to a solution of α-mangostin (1.0 equiv) in acetone. The reaction mixture was heated at reflux for 24 h. The reaction was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was diluted with ethyl acetate and then washed with saturated NaHCO3 (aq) and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to afford intermediates 1a to 1h. Excess diethyl amine was added to a solution of 1a in DMSO. The reaction was stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was diluted with ethyl acetate and washed with saturated NaHCO3 (aq) and brine. The organic phase was



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dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to obtain the pure product 2a. Compounds 2b to 2g were prepared from 1b to 1g using protocols similar to that for 2a. General Procedure B for the Synthesis of 3, 3a and 3b Potassium phthalimide (354 mg, 1.91 mmol) was added to a solution of 1c (500 mg, 0.73 mmol) in anhydrous DMF (5 mL). The resulting suspension was stirred at 90 °C for 12 h, then cooled to room temperature. The reaction mixture was diluted with ethyl acetate and washed with saturated NaHCO3 (aq) and brine. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography to afford the desired product 3. 3 (100 mg, 0.12 mmol) was dissolved in 4 mL of methylamine solution (33 wt. % in absolute ethanol). The reaction was refluxed at 70 ˚C for 7 h, then cooled to room temperature. After solvent removal under reduced pressure, the residue was diluted with dichloromethane and washed three times with saturated NaHCO3 (aq). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by preparative RP-HPLC with isocratic elution in 52% CH3OH/48% H2O (0.1% formic acid) to give product 3a. 1H-Pyrazole-1-carboxamidine hydrochloride (32 mg, 0.22 mmol) was added to a solution of 3a (46 mg, 0.08 mmol) in 3 mL of anhydrous DMF, followed by the addition of N,Ndiisopropylethylamine (DIEA, 40 µl). The reaction was stirred at room temperature overnight. Diethyl ether was added to the mixture after the reaction was complete. The resulting sticky suspension was centrifuged, and a yellow solid was collected. The solid was washed with diethyl ether and dried in vacuo. The crude product was purified by preparative RP-HPLC with isocratic elution with 52% CH3OH/48% H2O (0.1% formic acid) to give the pure product 3b.


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General Procedure C for the Synthesis of 4a – 4n and 5a – 5k Secondary amine was added to a solution of 1c (1.0 equiv) in DMSO. The reaction was stirred at room temperature for 4 h. The reaction mixture was diluted with ethyl acetate and washed with saturated NaHCO3 (aq) and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to afford the product. General Procedure D for Synthesis of 6a – 6c 1H-Imidazole (100 mg, 1.47 mmol) and potassium carbonate (101 mg, 0.74 mmol) were added to a solution of 1c (100 mg, 0.15 mmol) in acetone (8 mL). The reaction mixture was heated at reflux for 48 h, then cooled to room temperature. The solvent was removed under vacuum. The residue was diluted with ethyl acetate and washed three times with saturated brine. The organic phase was dried over anhydrous Na2SO4. The crude product was purified via silica gel column chromatography (EtOAc/MeOH/Et3N, 100/2/1, v/v) to afford product 5a. General Procedure E for the Synthesis of 8a to 8b The xanthone core 7 and intermediate 7a were synthesized according to the previously reported method.21 Briefly, 2,4-dihydroxybenzoic acid (1.0 equiv) and phloroglucinol (1.0 equiv) were dissolved in Eaton’s reagent (Phosphorus pentoxide, 7.7 wt. % in methanesulfonic acid). The reaction mixture was stirred at 80.0 ˚C for 1 h, then poured into ice. The solid was collected by filtration, washed with water and dried under vacuum. The crude product was purified by silica gel chromatography. Potassium carbonate (5.0 equiv) and dibromobutane (15.0 equiv) were added to a solution of 7a in acetone (1.0 equiv). The reaction mixture was stirred and refluxed overnight. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with sodium bicarbonate and brine. The organic layer was dried over anhydrous sodium sulfate, concentrated under vacuum, and



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purified by silica gel chromatography with petroleum ether/ethyl acetate (8:1, v/v) to obtain the intermediate 7a. Secondary amines were added to a solution of 7a (1.0 equiv) in DMSO. The reaction was stirred at room temperature for 4 h. The reaction mixture was diluted with ethyl acetate and washed with saturated NaHCO3 (aq) and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to afford products 8a and 8b. General Procedure F for the Synthesis of 10a to 10c 9 and intermediate 9a were synthesized according to the previously reported method.21 αMangostin (1.0 equiv) was dissolved in ethanol. Palladium on carbon (10 wt. % loading, matrix activated carbon support ) was added (15.0% w/w to α-mangostin) and the reaction mixture was stirred at room temperature for 2 h under a hydrogen atmosphere. The mixture was filtered and diluted with ethyl acetate, then purified by column chromatography on silica gel petroleum ether/ethyl acetate (6:1, v/v), affording the yellow solid 9 as the desired product. Potassium carbonate (5.0 equiv) and 1,4-dibromobutane (15.0 equiv) were added to a solution of 9 (1.0 equiv) in 8 mL of acetone. The reaction mixture was refluxed for 24 h. After the reaction was complete, the solvent was removed under reduced pressure. The oil residue was diluted with EtOAc, then washed twice with saturated brine and once with water. The organic phase was dried over anhydrous Na2SO4, then purified via silica gel column chromatography with petroleum ether/ethyl acetate (20:1, v/v) to obtain intermediate 9a. Secondary amines were added to a solution of 9a (1.0 equiv) in DMSO. The reaction was stirred at room temperature for 4 h. The reaction mixture was diluted with ethyl acetate, then washed with saturated NaHCO3 (aq) and brine. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography on silica gel to afford products 10a to 10c.


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General Procedure G for Synthesis of 12 α-Mangostin (500 mg, 1.22 mmol) was dissolved in 10 mL of acetone. Potassium carbonate (1.26g, 9.15 mmol) and 1,4-diiodobutane (2.41 mL, 18.30 mmol) were added. The reaction mixture was refluxed for 24 h. After the reaction was complete, the solvent was removed under reduced pressure. The oil residue was diluted with EtOAc and washed twice with saturated brine and once with water. The organic phase was dried over anhydrous Na2SO4, then purified via silica gel column chromatography (petroleum ether/EtOAc, 15/1, v/v) to obtain intermediate 11. Then, to a solution of 11 (100 mg, 0.11 mmol) in DMSO (4 mL), diethylamine (4 mL) was added. The mixture was stirred at room temperature for 4 h. After completion of the reaction, the mixture was diluted with 50 mL of ethyl acetate, then washed with aqueous NaHCO3 and saturated brine (each three times). The organic phase was dried over anhydrous Na2SO4 and concentrated under vacuum. The residual crude oil was purified via silica gel column chromatography (EtOAc/MeOH/Et3N, 100/2/1, v/v) to afford 12. Characterization of Compounds. 3,6-Bis(2-bromoethoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (1a) was prepared according to general procedure A from α-mangostin (300 mg, 0.73 mmol) and 1,2-dibromoethane (0.95 mL, 10.96 mmol). 1a was obtained as a light yellow solid (406 mg) in 89% yield. Mp 148–150 °C. 1H NMR (400 MHz, CDCl3) δ 13.45 (s, 1H, OH), 6.66 (s, 1H, Ar–H), 6.24 (s, 1H, Ar–H), 5.28 – 5.22 (m, 2H, 2×CH), 4.40 (t, 2H, CH2), 4.36 (t, 2H, CH2), 4.13 (d, J = 6.4, 2H, CH2), 3.85 (s, 3H, OCH3), 3.75 (t, 2H, CH2), 3.69 (t, 2H, CH2), 3.37 (d, J = 7.2, 2H, CH2), 1.85 (s, 3H, CH3), 1.81 (s, 3H, CH3), 1.68 (s, 6H, CH3).

13

C NMR (101 MHz, CDCl3) δ 182.12, 161.84, 160.28, 156.56, 155.17, 155.11,

144.24, 137.96, 132.07, 131.77, 123.19, 122.35, 112.77, 112.08, 104.47, 99.07, 89.46, 68.52,



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68.27, 61.36, 28.64, 28.57, 26.36, 26.05, 25.97, 21.62, 18.34, 18.03. MS (APCI): calcd for C28H33Br2O6 [M + H]+ 625.4, found 625.1. 3,6-Bis((8-bromooctyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (1f) was prepared according to general procedure A from α-mangostin (300 mg, 0.73 mmol) and 1,8-dibromoethane (2.02 mL, 10.96 mmol). 1f was obtained as a light yellow solid (451 mg) in 78% yield. Mp 73–75 °C. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.70 (s, 1H, Ar–H), 6.28 (s, 1H, Ar–H), 5.26 - 5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4,07 (t, 2H, CH2), 4.03 (t, 2H, CH2), 3.80(s, 3H, OCH3), 3.43 -3.40, (t, 4H, 2×CH2), 3.36 (d, 2H, CH2), 1.91-1.82 (m, 11H, 4×CH2, 1×CH3), 1.80 (s, 3H, CH3), 1.68 (s, 6H, 2×CH3). 1.53-1.38 (m, 16H, 8×CH2).

13

C NMR (101 MHz, CDCl3) δ 182.14, 162.98,

160.00, 157.59, 155.44, 155.31, 144.21, 137.32, 131.80, 131.44, 123.48, 122.68, 112.07, 111.64, 104.01, 98.84, 89.36, 68.91, 68.52, 60.97, 34.05 (2×CH2), 32.92, 32.88, 29.30, 29.24, 29.21, 29.05, 28.86, 28.79, 28.24, 28.20, 26.33, 26.14, 26.12, 26.05, 25.99, 21.62, 18.32, 17.97. MS (APCI): calcd for C40H57Br2O6 [M + H]+ 793.7, found 793.3. 3,6-Bis((10-bromodecyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (1g) was prepared according to general procedure A from α-mangostin (321 mg, 0.78 mmol) and 1,10-dibromoethane (3.52 g, 11.73 mmol). 1g was obtained as a light yellow oil (538 mg) in 81% yield. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.71 (s, 1H, Ar–H), 6.29 (s, 1H, Ar–H), 5.27 – 5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4,07 (t, 2H, CH2), 4.03 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.41 (t, 4H, 2×CH2), 3.36 (d, J = 7.2, 2H, CH2), 1.91-1.82 (s, 11H, 4×CH2, 1×CH3), 1.80 (s, 3H, CH3), 1.68 (s, 6H, CH3). 1.52-1.32 (m, 12×CH2, 24H).

13

C NMR (101 MHz, CDCl3) δ 182.16, 163.03, 160.01, 157.63, 155.46,

155.33, 144.23, 137.33, 131.81, 131.44, 123.49, 122.69, 112.07, 111.67, 104.02, 98.84, 89.36, 68.98, 68.59, 60.98, 34.13 (2×CH2), 32.97, 32.95, 29.60, 29.54, 29.50, 29.49, 29.44,


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29.39, 29.26, 29.09, 28.89, 28.87, 28.31, 28.29, 26.34, 26.22, 26.20, 26.06, 25.99, 21.63, 18.33, 17.97. MS (APCI): calcd for C44H65Br2O6 [M + H]+ 849.8, found 849.4. 3,6-Bis((12-bromododecyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (1h) was prepared according to general procedure A from α-mangostin (300 mg, 0.73 mmol) and 1,12-dibromoethane (2.02 mL, 10.96 mmol). 1h was obtained as a light yellow oil (482 mg) in 73% yield. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.71 (s, 1H, Ar–H), 6.29 (s, 1H, Ar–H), 5.27 – 5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4,07 (t, 2H, CH2), 4.03 (t, 2H, CH2), 3.80(s, 3H, OCH3), 3.41 (t, 4H, 2×CH2), 3.36 (d, 2H, J = 7.2, CH2), 1.93-1.82 (m, 11H, 4×CH2, 1×CH3), 1.80 (s, 3H, 1×CH3), 1.68 (s, 6H, 2×CH3). 1.531.29 (m, 32H, 16×CH2).

13

C NMR (101 MHz, CDCl3) δ 182.15, 163.04, 159.99, 157.63,

155.45, 155.32, 142.75, 137.30, 131.78, 131.43, 123.49, 122.70, 112.05, 111.65, 104.00, 98.84, 89.36, 68.99, 68.61, 60.96, 34.16 (2×CH2), 32.99 (2×CH2), 29.71, 29.65 (5×CH2), 29.56 (2×CH2), 29.48, 29.43, 29.27, 29.09, 28.91 (2×CH2), 28.31 (2×CH2), 26.34, 26.23, 26.21, 26.06, 25.99, 21.62, 18.33, 17.97. MS (APCI): calcd for C48H73Br2O6 [M + H]+ 905.9, found 905.4. 3,6-Bis(2-(diethylamino)ethoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9H-xanthen-9-one (2a) was prepared according to general procedure A from 1a (120 mg, 0.19 mmol) and diethylamine (4 mL). 2a was obtained as a light yellow oil (104 mg) in 89% yield. Purity: 98.5%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.74 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 5.26-5.22 (m, 2H, 2×CH), 4.16-4.11 (m, 6H, 3×CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J =7.2, 2H, CH2), 2.99-2.92 (m, 4H, 2×CH2), 2.69-2.63 (m, 8H, 4×CH2), 1.84 (3H, CH3), 1.80 (3H, CH3), 1.68 (6H, 2×CH3), 1.09 (t, 12H, 4×CH3). 13C NMR (101 MHz, CDCl3) δ 182.16, 162.77, 160.06, 157.46, 155.44, 155.29, 144.23, 137.44, 131.82, 131.55, 123.46, 122.66, 112.23, 111.72, 104.14, 99.05, 89.52, 67.89, 67.65, 61.00, 51.84, 51.75, 48.07



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(2×CH2), 48.02 (2×CH2), 26.33, 26.04, 25.95, 21.62, 18.32, 18.03, 12.19 (2×CH2), 12.17 (2×CH2). MS (ESI): calcd for C36H53N2O6 [M + H]+ 609.4, found 609.3. 3,6-Bis((8-(diethylamino)octyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (2f) was prepared according to general procedure A from 1f (112 mg, 0.14 mmol) and diethylamine (4 mL). 2f was obtained as a light yellow oil (88 mg) in 80% yield. Purity: 99.1%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.70 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J =6.4, 2H, CH2), 4.06 (t, 2H, CH2), 4.02 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J =7.2, 2H, CH2), 2.58-2.52 (m, 8H, 4×CH2), 2.45-2.41 (m, 4H, 2×CH2), 1.84-1.79 (m, 10H, 2×CH2, 2×CH3), 1.67 (s, 6H, 2×CH3), 1.511.29 (m, 20H, 10×CH2), 1.03 (t, 12H, 4×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.15,

163.02, 159.99, 157.62, 155.45, 155.32, 144.22, 137.29, 131.76, 131.41, 123.50, 122.69, 112.05, 111.64, 104.00, 98.84, 89.36, 68.97, 68.59, 60.96, 53.13 (2×CH2), 47.03 (4×CH2), 29.71, 29.65, 29.47, 29.41, 29.25, 29.08, 27.80, 27.78, 27.13 (2×CH2), 26.33, 26.19, 26.18, 26.04, 25.98, 21.62, 18.32, 17.96, 11.76 (4×CH3). MS (ESI): calcd for C48H77N2O6 [M + H]+ 777.6, found 778.2. 3,6-Bis((10-(diethylamino)decyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (2g) was prepared according to general procedure A from 1g (67 mg, 0.08 mmol) and diethylamine (4 mL). 2g was obtained as a light yellow oil (55 mg) in 83% yield. Purity: 99.5%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.70 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J =6.4, 2H, CH2), 4.06 (t, 2H, CH2), 4.02 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J =6.8, 2H, CH2), 2.56-2.51 (m, 8H, 4×CH2), 2.44-2.40 (m, 4H, 2×CH2), 1.92-1.79 (m, 10H, 2 × CH2, 2 × CH3), 1.67 (s, 6H, 2 × CH3), 1.53-1.30 (m, 28H, 14×CH2), 1.02 (t, 12H, 4×CH3). 13C NMR (101 MHz, CDCl3) δ 182.15, 163.04, 160.00, 157.64, 155.46, 155.32, 144.23, 137.29, 131.77, 131.42, 123.50, 122.70, 112.05, 111.66, 104.01, 98.84, 89.37, 69.00, 68.62, 60.96, 53.14 (2×CH2), 47.02 (4×CH2), 30 ACS Paragon Plus Environment

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29.76, 29.75, 29.71, 29.69 (2×CH2), 29.63, 29.49, 29.43, 29.27, 29.09, 27.87 (2×CH2), 27.11, 27.09, 26.34, 26.24, 26.21, 26.05, 25.98, 21.62, 18.32, 17.96, 11.75 (4×CH3). MS (ESI): calcd for C52H85N2O6 [M + H]+ 833.6, found 834.3. 3,6-Bis((12-(diethylamino)dodecyl)oxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (2h) was prepared according to general procedure A from 1h (105 mg, 0.116 mmol) and diethylamine (4 mL). 2h was obtained as a light yellow oil (89 mg) in 89% yield. Purity: 98.8%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.71 (s, 1H, ArH), 6.29 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J =6.4, 2H, CH2), 4.07 (t, 2H, CH2), 4.02 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J =6.8, 2H, CH2), 2.60-2.55 (m, 8H, 4×CH2), 2.47-2.43 (m, 4H, 2×CH2), 1.91-1.80 (m, 10H, 2×CH2, 2×CH3), 1.67, (s, 6H, 2×CH3) 1.53-1.28 (m, 36H, 18×CH2), 1.05 (t, 12H, 4×CH3).13C NMR (101 MHz, CDCl3) δ 182.15, 163.04, 159.98, 157.63, 155.45, 155.32, 144.22, 137.28, 131.76, 131.41, 123.50, 122.69, 112.04, 111.65, 103.99, 98.83, 89.36, 69.00, 68.62, 60.96, 53.06 (2×CH2), 46.98 (4×CH2), 29.75 (4×CH2), 29.72 (2×CH2), 29.70, 29.66, 29.50, 29.44, 29.27, 29.09, 27.85 (2×CH2), 26.91, 26.89, 26.33, 26.24, 26.21, 26.04, 25.97, 21.62, 18.31, 17.96, 11.60 (4×CH3). MS (ESI): calcd for C56H93N2O6 [M + H]+ 889.7, found 890.4. 2,2'-(((1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-xanthene-3,6diyl)bis(oxy))bis(butane-4,1-diyl))bis(isoindoline-1,3-dione) (3) was prepared according to general procedure B from 1c (500 mg, 0.73 mmol) and potassium phthalimide (354 mg, 1.91 mmol). 3 was obtained as a light yellow solid (490 mg) in 82% yield. Mp 107–109 °C. Purity: 97.1%. 1H NMR (400 MHz, CDCl3) δ 13.46 (s, 1H, OH), 7.86-7.82 (m, 4H, 4×Ar-H), 7.72-7.69 (m, 4H, 4×Ar-H), 6.70 (s, 1H, Ar-H), 6.27 (s, 1H, Ar-H), 5.23-5.19 (m, 2H, 2×CH), 4.12-4.07 (m, 6H, 3×CH2), 3.80-3.74 (m, 7H, 1×OCH3, 2×CH2), 3.33 (d, 2H, J = 6.8, CH2), 1.95-1.91 (8H, 4×CH2), 1.84(s, 3H, CH3), 1.76 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.64 (s, 3H, CH3).

13

C NMR (101 MHz, CDCl3) δ 182.09, 168.53 (4×CO), 162.68, 159.99, 157.34,



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Page 32 of 84

155.34, 155.21, 144.18, 137.38, 134.12 (2×Ar-H), 134.06 (2×Ar-H), 132.20 (2×Ar-H), 132.19 (2×Ar-H), 131.78, 131.47, 123.43, 123.37 (2×Ar-H), 123.33 (2×Ar-H), 122.60, 112.18, 111.67, 104.06, 98.92, 89.39, 68.09, 67.68, 61.06, 37.64, 37.58, 26.59, 26.45, 26.30, 26.03, 25.89, 25.39, 25.36, 21.57, 18.30, 17.93. MS (APCI): calcd for C48H49N2O10 [M + H]+ 813.3, found 813.2. 3,6-Bis(4-aminobutoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9H-xanthen-9one (3a) was prepared according to general procedure B from 3 (100 mg, 0.12 mmol) and methylamine solution (10 mL, 33 wt. % in absolute ethanol). 3a was obtained as a light yellow solid (39 mg, 58%) in 82% yield. Mp 136–138 °C. Purity: 98.3%. 1H NMR (400 MHz, MeOD) δ 6.89 (s, 1H, Ar-H), 6.44 (s, 1H, Ar-H), 5.24-5.18 (m, 2H, 2×CH), 4.19 (t, 2H, CH2), 4.14 (t, 2H, CH2), 4.10 (d, 2H, CH2, J = 6.4), 3.79 (s, 3H, CH3), 3.33 (d, 2H, CH2), 3.09-3.02 (m, 4H, 2×CH2), 2.02-1.88 (m, 8H, 4×CH2) 1.84 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.67 (s, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 183.26, 164.09, 160.67, 158.85, 156.76, 156.59, 145.48, 138.08, 132.06, 132.03, 124.81, 123.69, 112.93, 112.54, 104.72, 100.30, 90.69, 69.50, 69.00, 61.46, 40.57, 40.55, 27.33, 27.17, 26.99, 26.02, 25.95, 25.88, 25.74, 22.31, 18.34, 18.07. MS (ESI): calcd for C32H45N2O6 [M + H]+ 553.3, found 553.2, HPLC tR = 12.5 min. 3,6-Bis((4-guanidinyl)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-enyl)-9Hxanthen-9-one (3b) was prepared according to general procedure B from 3a (46 mg, 0.084 mmol) and pyrazole-1-carboxamidine hydrochloride (32 mg, 0.22 mmol). 3b was obtained as a light yellow oil (40 mg, 74%) in 82% yield. Purity: 99.4%. 1H NMR (400 MHz, DMSO-d6) δ = 13.52 (1H, s, OH), 8.43 (br, 2H, NH), 7.84 (br, 6H, 2×NH, 2×NH2), 7.03 (1H, s, Ar-H), 6.54(1H, s, Ar-H),5.15-5.14 (m, 2H, 2×CH), 4.18-4.12 (m, 4H, 2×CH2), 4.01 (2H, d, J = 6.4, CH2), 3.72 (s, 3H, OCH3), 3.23 (d, J = 7.2, 2H, CH2), 3.17-3.15 (m, 4H, 2×CH2), 1.83-1.62 (m, 20H, 4×CH3, 4×CH2).

13

C NMR (101 MHz, DMSO-d6) δ = 181.47, 162.55, 158.80,


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157.49, 156.99 (2×C=NH), 154.84, 154.70, 143.80, 135.72, 130.70, 130.57, 123.35, 122.08, 110.72, 110.57, 102.84, 99.53, 89.94, 68.31, 67.93, 60.37, 40.28, 40.24, 25.72, 25.57 (2×CH2), 25.53, 25.48, 25.26, 25.20, 20.99, 17.97, 17.65. MS (ESI): calcd for C34H49N6O6 [M + H]+ 637.4, found 637.3, HPLC tR = 11.5 min. 3,6-Bis(4-(dimethylamino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9H-xanthen-9-one (4a) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and dimethylamine (4 mL, 2.0 M in methanol). 4a was obtained as a light yellow oil (53 mg) in 59% yield. Purity: 99.4%. 1H NMR (400 MHz, CDCl3) δ = 13.49 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.25-5.22 (m, 2H, 2×CH), 4.14-4.04 (m, 6H, CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J = 6.4, 2H, CH2), 2.41-2.36 (m, 4H, 2×CH2), 2.27 (s, 12H, 4×CH3), 1.95 -1.91 (m, 2H, CH2), 1.90-1.86 (m, 2H, CH2), 1.84 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.74-1.70 (m, 4H, 2×CH2), 1.67 (s, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ

182.35, 163.09, 160.22, 157.74, 155.64, 155.51, 144.41, 137.56, 132.01, 131.69, 123.66, 122.87, 112.33, 111.85, 104.26, 99.10, 89.58, 68.98, 68.56, 61.19, 59.59, 59.54, 45.68 (2×CH3), 45.64 (2×CH3), 27.34, 27.19, 26.53, 26.24, 26.16, 24.51, 24.43, 21.81, 18.52, 18.18. MS (ESI): calcd for C36H53N2O6 [M + H]+ 609.4, found 609.2. 3,6-Bis(4-(bis(2-hydroxyethyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut2-en-1-yl)-9H-xanthen-9-one (4b) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and diethanolamine (4 mL). 4b was obtained as a light yellow oil (80 mg) in 75% yield. Purity: 99.1%. 1H NMR (400 MHz, DMSO-d6) δ 13.57 (s, 1H, OH), 7.07 (s, 1H, Ar-H), 6.58 (s, 1H, Ar-H), 5.21-5.18 (m, 2H, 2×CH), 4.21 (t, 2H, CH2), 4.15 (t, 2H, CH2), 4.07 (d, J = 6.8, 2H, CH2), 3.77 (s, 3H, 1×OCH3), 3.54-3.50 (m, 8H, 4×OCH2), 3.29 (d, J = 7.2, 2H, CH2), 2.71-2.65 (m, 12H, 6×CH2), 1.87-1.77 (m, 10H, 2×CH2, 2×CH3), 1.691.66 (m, 10H, 2×CH2, 2×CH3).

13

C NMR (101 MHz, DMSO) δ 182.39, 163.57, 159.70,

158.48, 155.77, 155.63, 144.71, 136.59, 131.56, 131.45, 124.33, 123.06, 111.59, 111.46,



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103.72, 100.37, 90.78, 69.62, 69.25, 61.24, 59.71 (2×CH2), 59.65 (2×CH2), 57.26 (2×CH2), 57.24 (2×CH2), 55.10, 55.03, 27.23, 27.06, 26.46, 26.50, 26.40, 23.85 (2×CH2), 21.93, 18.89, 18.56. MS (ESI): calcd for C40H61N2O10 [M + H]+ 729.4, found 729.3. 3,6-Bis(4-(ethyl(methyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (4c) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-ethylmethylamine (4 mL). 4c was obtained as a light yellow oil (73 mg) in 78% yield. Purity: 95.4%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H, ArH), 6.28 (s, 1H, Ar-H), 5.25-5.21 (m, 2H, 2×CH), 4.13-4.04 (m, 6H, 3×CH2), 3.79 (s, 3H, 1×OCH3), 3.35 (d, J = 7.2, 2H, CH2), 2.51-2.44 (m, 8H, 4×CH2), 2.26 (s, 6H, 2×CH3), 1.941.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, CH3), 1.77-1.69 (m, 4H, 2×CH2), 1.67 (s, 6H, 2×CH3), 1.09 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.13, 162.86, 160.00, 157.51, 155.43, 155.29, 144.20, 137.33, 131.77, 131.45, 123.47, 122.67, 112.10, 111.62, 104.04, 98.90, 89.37, 68.80, 68.36, 60.97, 56.87, 56.83, 51.49, 51.42, 41.52, 41.48, 27.26, 27.11, 26.31, 26.03, 25.94, 23.89, 23.76, 21.60, 18.30, 17.97, 12.12, 12.12. MS (ESI): calcd for C38H57N2O6 [M + H]+ 637.4, found 637.5. 1-Hydroxy-7-methoxy-3,6-bis(4-(methyl(propyl)amino)butoxy)-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (4d) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-methylpropylamine (4 mL). 4d was obtained as a light yellow oil (85 mg) in 85% yield. Purity: 96.0%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.26-5.22 (m, 2H, 2×CH), 4.13 (d, J = 6.8, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 7.2, 2H, CH2), 2.48 (t, 4H, 2×CH2), 2.35 (t, 4H, 2×CH2), 2.26 (s, 6H, 2×CH3), 1.94-1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s 3H, CH3), 1.74-1.67 (m, 10H, 2×CH2, 2×CH3), 1.56-1.46 (m, 4H, 2×CH2), 0.90 (t, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ 181.33, 162.09, 159.20, 156.73, 154.63, 154.49,

143.41, 136.52, 130.97, 130.64, 122.68, 121.87, 111.29, 110.82, 103.23, 98.10, 88.57, 68.01,


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67.60, 60.16, 58.91, 58.83, 56.44, 56.39, 41.27, 41.21, 26.46, 26.29, 25.52, 25.23, 25.15, 22.99, 22.87, 20.81, 19.46 (2×CH2), 17.50, 17.16, 11.22 (2×CH2). MS (ESI): calcd for C40H61N2O6 [M + H]+ 665.5, found 665.7. 3,6-Bis(4-(ethyl(2-hydroxyethyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3methylbut-2-en-1-yl)-9H-xanthen-9-one (4e) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and 2-(ethylamino)ethanol (4 mL). 4e was obtained as a light yellow oil (79 mg) in 79% yield. Purity: 98.8%. 1H NMR (400 MHz, CDCl3) δ 13.46 (s, 1H, OH), 6.69 (s, 1H, Ar-H), 6.27 (s, 1H, Ar-H), 5.23-5.16 (m, 2H, 2×CH), 4.11-4.07 (m, 6H, 3×CH2), 3.90 (s, 4H, 2×CH2), 3.76 (s, 3H, 1×OCH3), 3.31 (d, J = 6.8, 2H, CH2), 3.14-3.07 (m, 12H, 6×CH2), 1.96-1.93 (m, 8H, 4×CH2), 1.82 (s, 3H, CH3), 1.77 (s, 3H, CH3), 1.66 (s, 3H, CH3), 1.65 (s, 3H, CH3), 1.30 (t, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.08,

162.40, 160.05, 157.13, 155.37, 155.23, 144.13, 137.56, 131.89, 131.61, 123.31, 122.54, 112.35, 111.47, 104.18, 99.10, 89.45, 68.11, 67.51, 61.03, 56.89, 56.87, 55.80, 55.78, 53.23, 53.04, 48.62, 48.52, 26.68, 26.52, 26.29, 26.01, 25.94, 21.60, 21.24, 20.85, 18.30, 17.99, 8.91, 8.85. MS (ESI): calcd for C40H61N2O8 [M + H]+ 697.4, found 697.7. 3,6-Bis(4-(butyl(methyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (4f) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-butylmethylamine (4 mL). 4f was obtained as a light yellow oil (89 mg) in 87% yield. Purity: 96.9%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.72 (s, 1H, ArH), 6.29 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4.10 (t, 2H, CH2), 4.06 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 7.2, 2H, CH2), 2.43-2.39 (m, 4H, 2×CH2), 2.35-2.32 (m, 4H, 2×CH2), 2.22 (s, 6H, 2×CH3), 1.94-1.88 (m, 4H, 2×CH2), 1.85 (s, 3H, 1×CH3), 1.80 (s, 3H, 1×CH3), 1.71-1.67 (m, 10H, 2×CH2, 2×CH3), 1.49- 1.42 (m, 4H, 2×CH2), 1.36-1.25 (m, 4H, 2×CH2), 0.93-0.90 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.35, 163.14, 160.22, 157.77, 155.65, 155.52, 144.43, 137.54, 131.99, 131.66, 123.68,



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122.87, 112.30, 111.86, 104.24, 99.09, 89.58, 69.09, 68.69, 61.18, 57.97, 57.93, 57.76, 57.67, 42.58, 42.55, 29.86, 29.83, 27.52, 27.35, 26.53, 26.24, 26.17, 24.28, 24.20, 21.82, 21.08 (2×CH2), 18.52, 18.18, 14.41 (2×CH3). MS (ESI): calcd for C42H65N2O6 [M + H]+ 693.5, found 693.8. 3,6-Bis(4-(ethyl(propyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (4g) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-ethyl-N-propylamine (4 mL). 4g was obtained as a light yellow oil (83 mg) in 81% yield. Purity: 98.7%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 7.2, 2H, CH2), 2.55-2.48 (m, 8H, 2×CH2), 2.41-2.38 (m, 4H, 2×CH2), 1.93-1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, CH3), 1.69-1.65 (m, 10H, 2×CH2, 2×CH3), 1.50-1.44 (m, 4H, 2×CH2), 1.02 (t, 6H, 2×CH3), 0.88 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.35, 163.15, 160.21, 157.78, 155.65, 155.52, 144.43, 137.53, 131.98, 131.64, 123.69, 122.87, 112.29, 111.85, 104.24, 99.10, 89.58, 69.14, 68.73, 61.16, 55.91, 55.89, 53.50, 53.45, 47.81, 47.77, 27.59, 27.41, 26.53, 26.24, 26.16, 24.11, 23.96, 21.82, 20.54, 20.52, 18.51, 18.17, 12.33 (2×CH3), 11.99 (2×CH3). MS (ESI): calcd for C42H65N2O6 [M + H]+ 693.5, found 693.4. 3,6-Bis(4-(ethyl(isopropyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (4h) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-ethylisopropylamine (4 mL). 4h was obtained as a light yellow oil (85 mg) in 83% yield. Purity: 98.4%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.25-5.24 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 7.2, 2H, CH2), 3.00-2.94 (m, 2H, 2×CH), 2.52-2.44 (m, 8H, 4×CH2), 1.95-1.86 (m, 4H, 2×CH2), 1.84 (s, 3H, 1×CH3), 1.80 (s, 3H, 1×CH3), 1.67-1.61 (m, 10H, 2×CH2, 2×CH3), 1.06-0.99 (s, 18H, 6×CH3).

13

C NMR


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(101 MHz, CDCl3) δ 182.15, 162.98, 159.99, 157.60, 155.45, 155.31, 144.22, 137.28, 131.76, 131.43, 123.50, 122.68, 112.06, 111.64, 104.01, 98.88, 89.38, 69.04, 68.62, 60.96, 50.25, 50.21, 49.28, 49.20, 43.96, 43.90, 27.38, 27.19, 26.33, 26.04, 25.96, 25.63, 25.52, 21.62, 18.47 (2×CH3), 18.46 (2×CH3), 18.31, 17.97, 14.24, 14.23. MS (ESI): calcd for C42H65N2O6 [M + H]+ 693.5, found 693.5. 1-Hydroxy-7-methoxy-3,6-bis(4-(methyl(pentyl)amino)butoxy)-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (4i) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-methylpropylamine (4 mL). 4i was obtained as a light yellow oil (91 mg) in 86% yield. Purity: 97.4%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.8, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 6.8, 2H, CH2), 2.43-2.38 (m, 4H, 2×CH2), 2.35-2.31 (m, 4H, 2×CH2), 2.22 (s, 6H, 2×CH3), 1.86-1.79 (m, 10H, 2×CH2, 2×CH3), 1.71-1.67 (m, 10H, 2×CH2, 2×CH3), 1.49-1.30 (m, 4H, 2×CH2), 1.30-1.26 (m, 8H, 4×CH2), 0.88 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.35, 163.14, 160.21, 157.77, 155.65, 155.51, 144.42, 137.53, 131.98, 131.65, 123.69, 122.87, 112.30, 111.86, 104.24, 99.09, 89.58, 69.08, 68.69, 61.17, 58.25, 58.20, 57.75, 57.67, 42.56, 42.54, 30.14 (2×CH2), 27.52, 27.36 (2×CH2), 27.32, 26.24, 26.16, 24.26, 24.53, 24.18, 23.00, 22.99, 21.82, 18.51, 18.17, 14.40 (2×CH3). MS (ESI): calcd for C44H69N2O6 [M + H]+ 721.5, found 721.4. 6-Bis(4-(butyl(ethyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9H-xanthen-9-one (4j) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-ethylbutylamine (4 mL). 4j was obtained as a light yellow oil (100 mg) in 95% yield. Purity: 96.8%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.27-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.8, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (d, J = 6.8, 2H, CH2), 2.56-2.48 (m, 8H, 4×CH2), 2.45-2.41 (m, 4H, 2×CH2), 1.93-1.80 (m, 10H, 2×CH2, 2×CH3), 1.69-1.62 (m, 10H,



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2×CH2, 2×CH3), 1.47 -1.39 (m, 4H, 2×CH2), 1.35-1.25 (m, 4H, 2×CH2), 1.02 (t, 6H, 2×CH3), 0.91 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.36, 163.16, 160.22, 157.79, 155.66, 155.52, 144.43, 137.54, 132.00, 131.66, 123.68, 122.87, 112.30, 111.86, 104.24, 99.10, 89.58, 69.15, 68.74, 61.17, 53.61, 53.59, 53.50, 53.46, 47.79, 47.75, 29.56, 29.53, 27.60, 27.43, 26.54, 26.25, 26.17, 24.12, 23.96, 21.83, 21.14 (2×CH2), 18.52, 18.17, 14.43 (2×CH2), 12.02, 12.01. MS (ESI): calcd for C44H69N2O6 [M + H]+ 721.5, found 721.8. 3,6-Bis(4-(dipropylamino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9H-xanthen-9-one (4k) was prepared according to general procedure C from 1c (200 mg, 0.29 mmol) and dipropylamine (4 mL). 4k was obtained as a light yellow oil (181 mg) in 86% yield. Purity: 97.9%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.30 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.14 (d, J = 6.8, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.37 (d, J = 6.8, 2H, CH2), 2.51-2.47 (m, 4H, 2×CH2), 2.40-2.36 (m, 8H, 4×CH2), 1.93-1.80 (m, 10H, 2×CH2, 2×CH3), 1.70-1.61 (m, 10H, 2×CH2, 2×CH3), 1.50-1.43 (m, 8H, 4×CH2), 0.90-0.86 (m, 12H, 4×CH3).

13

C NMR (101 MHz,

CDCl3) δ 182.36, 163.19, 160.22, 157.81, 155.67, 155.53, 144.43, 137.53, 132.00, 131.65, 123.69, 122.86, 112.29, 111.87, 104.24, 99.09, 89.59, 69.18, 68.79, 61.16, 56.61 (2×CH2,) 56.58 (2×CH2), 54.22, 54.15, 27.57, 27.38, 26.54, 26.25, 26.18, 24.25, 24.12, 21.83, 20.66 (2×CH2), 20.63 (2×CH2), 18.52, 18.17, 12.32 (4×CH3). MS (ESI): calcd for C44H69N2O6 [M + H]+ 721.5, found 721.4. 3,6-Bis(4-(hexyl(methyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (4l) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-hexylmethylamine (4 mL). 4l was obtained as a light yellow oil (100 mg) in 91% yield. Purity: 98.2%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, 1×OCH3), 3.36 (J = 6.4, 2H, CH2), 2.43-2.39 (m,


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4H, 2×CH2), 2.33-2.31 (m, 4H, 2×CH2), 2.22 (m, 6H, 2×CH3), 1.94-1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, CH3), 1.71-1.67 (m, 10H, 2×CH2, 2×CH3), 1.46-1.44 (m, 4H, 2×CH2), 1.37-1.28 (m, 12H, 6×CH2), 0.88 (t, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.32,

163.12, 160.19, 157.75, 155.62, 155.49, 144.41, 137.49, 131.93, 131.61, 123.69, 122.88, 112.27, 111.83, 104.21, 99.07, 89.56, 69.06, 68.66, 61.14, 58.26, 58.21, 57.71, 57.63, 42.54, 42.51, 32.19, 32.18, 27.61(4×CH3), 27.51, 27.34, 26.51, 26.23, 26.15, 24.23, 24.14, 22.98 (2×CH2), 21.81, 18.50, 18.16, 14.39 (2×CH3). MS (ESI): calcd for C46H73N2O6 [M + H]+ 749.5, found 749.4. 3,6-Bis(4-(dibutylamino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)9H-xanthen-9-one (4m) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and dibutylamine (4 mL). 4m was obtained as a light yellow oil (102 mg) in 90% yield. Purity: 97.0%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.29 (S, 1H, Ar-H), 5.26-5.23 (m, 2H, 2×CH), 4.13 (d, J = 6.4, 2H, CH2),4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.80 (s, 3H, OCH3), 3.36 (d, J = 6.4, 2H, CH2), 2.52-2.48 (m, 4H, 2×CH2), 2.44-2.41 (m, 8H, 4×CH2), 1.93-1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, 1×CH3), 1.711.63 (m, 10H, 2×CH2, 2×CH3), 1.46,-1.39 (m, 8H, 4×CH2), 1.35-1.25 (m, 8H, 4×CH2), 0.930.89 (t, 12H, 4×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.34, 163.14, 160.20, 157.77,

155.64, 155.50, 144.41, 137.50, 131.96, 131.62, 112.27, 111.83, 104.22, 99.09, 89.58, 69.14, 68.74, 61.14, 54.20 (2×CH2), 54.15 (2×CH2), 54.09, 54.05, 29.49 (4×CH2), 27.56, 27.38, 26.52, 26.23, 26.17, 24.11, 23.94, 21.82, 21.09 (2×CH2), 18.50, 18.16, 14.42 (2×CH2). MS (ESI): calcd for C48H77N2O6 [M + H]+ 776.6, found 777.4. 3,6-Bis(4-(benzyl(methyl)amino)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (4n) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and N-benzylmethylamine (4 mL). 4n was obtained as a light yellow oil (101 mg) in 91% yield. Purity: 97.6%. 1H NMR (400 MHz, CDCl3) δ 13.50 (s, 1H, OH), 7.34-7.28



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(m, 8H, 8×Ar-H), 7.25-7.23 (m, 2H, 2×Ar-H), 6.69 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.275.23 (m, 2H, 2×CH), 4.14 (d, J = 6.4, 2H, CH2), 4.07 (t, 2H, CH2), 4.03 (t, 2H, CH2), 3.79 (s, 3H, 1×OCH3), 3.50 (s, 4H, 2×CH2), 3.37 (d, J = 7.2, 2H, CH2), 2.47-2.43 (m, 4H, 2×CH2), 2.22 (s, 3H, CH3), 2.21 (s, 3H, CH3), 1.96-1.87 (m, 4H, 2×CH2), 1.85 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.78,-1.73 (m, 4H, 2×CH2), 1.68 (s, 3H, CH3), 1.67 (s, 3H, CH3). 13C NMR (101 MHz, CDCl3) δ 182.36, 163.16, 160.22, 157.78, 155.65, 155.52, 144.43, 139.56, 139.50, 137.53, 131.99, 131.68, 129.35 (4×Ar-H), 128.57 (2×Ar-H), 128.55 (2×Ar-H), 127.31, 127.26, 123.69, 122.89, 112.30, 111.86, 104.24, 99.11, 89.60, 68.99, 68.60, 62.77, 62.71, 61.17, 57.13, 56.99, 42.56, 42.51, 27.28, 27.09, 26.54, 26.25, 26.17, 24.23, 24.18, 21.83, 18.53, 18.19. MS (ESI): calcd for C48H61N2O6 [M + H]+ 761.5, found 761.7. 3,6-Bis(4-((S)-3-(dimethylamino)pyrrolidin-1-yl)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3methylbut-2-en-1-yl)-9H-xanthen-9-one (5b) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and (S)-(−)-3-(dimethylamino)pyrrolidine (4 mL). 5b was obtained as a brown oil (76 mg) in 69% yield. Purity: 95.9%. 1H NMR (400 MHz, CDCl3) δ 13.48 (s, 1H, OH), 6.71 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.22-5.21 (m, 2H, 2×CH), 4.124.04 (m, 6H, 3×CH2), 3.78 (s, 3H, 1×OCH3), 3.34 (d, J = 6.8, 2H, CH2), 3.28 – 3.26 (m, 2H, 2×CH), 3.04-2.71 (m, 12H, 6×CH2), 2.43 (m, 12H, 4×CH3), 2.15-2.13 (m,2H, CH2),-1.78 (16H, 6×CH2, 4×CH3), 1.66 (s, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.12, 162.69, 160.04, 157.37, 155.42, 155.28, 144.18, 137.45, 131.85, 131.51, 123.39, 122.61, 112.22, 111.56, 104.11, 98.98, 89.40, 68.45, 68.01, 64.26, 64.24, 61.01, 56.35, 56.28, 55.42, 55.37, 52.60, 52.56, 42.26 (2×CH2), 42.22 (2×CH2), 27.06, 27.04, 26.99, 26.83, 26.31, 26.03, 25.97, 24.28, 24.22, 21.61, 18.31, 17.98. MS (ESI): calcd for C44H67N4O6 [M + H]+ 747.5, found 747.2. 1-Hydroxy-3,6-bis(4-(4-hydroxypiperidin-1-yl)butoxy)-7-methoxy-2,8-bis(3-methylbut-2en-1-yl)-9H-xanthen-9-one (5d) was prepared according to general procedure C from 1c (100


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mg, 0.15 mmol) and 4-hydroxypiperidine (300 mg). 5d was obtained as a brown oil (75 mg) in 71% yield. Purity: 95.4%. 1H NMR (400 MHz, MeOD) δ 6.74 (s, 1H, Ar-H), 6.32 (s, 1H, Ar-H), 5.23 (t, 1H, CH), 5.17 (t, 1H, CH), 4.13-4.05 (m, 6H, 3×CH2), 3.97-3.94 (m, 2H, 2×CH), 3.78 (s, 3H, 1×OCH3), 3.39-3.38 (m, 4H, 2×CH2), 3.25 (d, 2H, CH2), 3.17-3.12 (m, 8H, 4×CH2), 2.06-1.79 (m, 22H, 8×CH2, 2×CH3), 1.69 (s, 3H, CH3), 1.67 (s, 3H, CH3). 13C NMR (101 MHz, MeOD) δ 183.10, 163.92, 160.51, 158.69, 156.55, 156.37, 145.36, 137.86, 131.99 (2×CH), 124.92, 123.82, 112.80, 112.34, 104.58, 100.26, 90.67, 69.35, 68.87, 63.81 (2×CH2), 61.48, 57.60, 57.59, 50.40 (2×CH2), 50.36(2×CH2), 32.00 (4×CH2), 27.56, 27.38, 27.03, 26.09, 26.00, 22.52, 22.42, 22.37, 18.41, 18.16. MS (ESI): calcd for C42H61N2O8 [M + H]+ 721.4, found 721.4. 1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-3,6-bis(4-thiomorpholinobutoxy)9H-xanthen-9-one (5f) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and thiomorpholine (4 mL). 5f was obtained as a brown oil (69 mg) in 65% yield. Purity: 99.5%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.79 (s, 1H, Ar-H), 6.36 (s, 1H, Ar-H), 5.25-5.23 (m, 2H, 2×CH), 4.14-4.04 (m, 6H, 3×CH2), 3.79 (s, 3H, 1×OCH3), 3.35 (d, J = 7.2, 2H, CH2), 2.72-2.70 (m, 16H, 8×CH2), 2.47-2.42 (m, 4H, 2×CH2), 1.95-1.79 (m, 10H, 2×CH2, 2×CH3), 1.70-1.67 (m, 10H, 2×CH2, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ

182.13, 162.87, 160.02, 157.49, 155.45, 155.30, 144.21, 137.40, 131.83, 131.49, 123.44, 122.67, 112.11, 111.63, 104.03, 98.99, 89.45, 68.72, 68.31, 60.98, 58.82, 58.65, 55.19 (2×CH2), 55.17 (2×CH2), 28.13 (2×CH2), 28.11 (2×CH2), 27.05, 26.77, 26.33, 26.05, 25.98, 23.08, 23.07, 21.62, 18.32, 17.99. MS (ESI): calcd for C40H57N2O6S2 [M + H]+ 725.4, found 724.8. 1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-3,6-bis(4-(piperazin-1-yl)butoxy)9H-xanthen-9-one (5g) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and piperazine (4 mL). 5g was obtained as a light yellow oil (74 mg) in 73%



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yield. Purity: 99.5%. 1H NMR (400 MHz, CDCl3) δ 13.48 (s, 1H, OH), 6.72 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.23-5.22 (m, 2H, 2×CH), 4.13-4.05 (m, 6H, 3×CH2), 3.79 (s, 3H, 1×OCH3), 3.60-3.56 (m, 4H, 2×CH2), 3.41-3.38 (m, 4H, 2×CH2), 3.35 (d, J = 7.2, 2H, CH2), 2.50-2.43 (m, 12H, 6×CH2), 1.96-1.87 (m, 4H, 2×CH2), 1.84 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.76-1.70 (m, 4H, 2×CH2), 1.67 (s, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.10,

162.82, 160.05, 157.46, 155.41, 155.27, 144.21, 137.48, 131.87, 131.50, 123.38, 122.64, 112.17, 111.64, 104.06, 98.87, 89.30, 68.64, 68.21, 60.98, 57.99, 57.86, 53.64, 53.60, 52.46 (2×CH2), 45.65 (2×CH2), 40.01 (2×CH2), 27.09, 26.86, 26.32, 26.03, 25.97, 23.27 (2×CH2), 21.61, 18.32, 17.98. MS (ESI): calcd for C40H59N4O6 [M + H]+ 691.4, found 691.4. 1-Hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-3,6-bis(4-(4-methylpiperazin-1yl)butoxy)-9H-xanthen-9-one (5h) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and 1-methylpiperazine (4 mL). 5h was obtained as a light yellow oil (83 mg) in 79% yield. Purity: 95.1%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-H), 5.25-5.21 (m, 2H, 2×CH), 4.12 (d, J = 6.8, 2H, CH2), 4.09 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.79 (s, 3H, 1×OCH3), 3.35 (d, J = 6.8, 2H, CH2), 2.50-2.42 (20H, 10×CH2), 2.30 (s, 6H, 2×CH3), 1.94 -1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, CH3), 1.75-1.71 (m, 4H, 2×CH2), 1.67 (s, 6H, 2×CH3).

13

C NMR (101 MHz, CDCl3) δ 181.33,

162.07, 159.23, 156.72, 154.63, 154.49, 143.42, 136.59, 131.01, 130.68, 122.64, 121.86, 111.33, 110.85, 103.25, 98.09, 88.55, 68.00, 67.57, 60.19, 57.41, 57.32, 54.36 (4×CH2), 52.35 (4×CH2), 45.26 (2×CH2), 26.48, 26.32, 25.53, 25.24, 25.16, 22.74, 22.71, 20.81, 17.52, 17.18. (ESI): calcd for C42H63N4O6 [M + H]+ 719.5, found 719.4. 3,6-Bis(4-(4-ethylpiperazin-1-yl)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en1-yl)-9H-xanthen-9-one (5i) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and 1-ethylpiperazine (4 mL). 5i was obtained as a light yellow oil (85 mg) in 78% yield. Purity: 96.0%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H,


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Ar-H), 6.28 (s, 1H, Ar-H), 5.25-5.22 (m, 2H, 2×CH), 4.12 (d, J = 6.8, 2H, CH2), 4.09 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.79 (s, 3H, OCH3), 3.35 (d, J = 6.8, 2H, CH2), 2.54-2.34 (m, 24H, 12×CH2), 1.94-1.84 (m, 7H, 2×CH2, 1×CH3), 1.79 (s, 3H, CH3), 1.76-1.65 (m, 10H, 2×CH2, 2×CH3), 1.10 (t, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ 182.33, 163.07, 160.22, 157.72, 155.63, 155.49, 144.41, 137.58, 132.01, 131.67, 123.64, 122.86, 112.32, 111.85, 104.25, 99.09, 89.56, 69.00, 68.58, 61.19, 58.45, 58.37, 53.33 (4×CH2), 53.01 (4×CH2), 52.62 (2×CH2), 27.49, 27.33, 26.52, 26.24, 26.16, 23.74, 23.71, 21.81, 18.51, 18.18, 12.15, 12.13. MS (ESI): calcd for C42H63N4O6 [M + H]+ 747.5, found 747.4. 3,6-Bis(4-(4-(2-(dimethylamino)ethyl)piperazin-1-yl)butoxy)-1-hydroxy-7-methoxy-2,8bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (5j) was prepared according to the general procedure C from 1c (100 mg, 0.15 mmol) and 1-Boc-piperazine (546 mg). 5j was obtained as a light yellow oil (75 mg) in 62% yield. Purity: 97.3%. 1H NMR (400 MHz, MeOD) δ 6.71(s, 1H, Ar-H), 6.28(s, 1H, Ar-H), 5.25 (t, 1H, 1×CH), 5.17 (t, 1H, 1×CH), 4.09-4.05 (m, 6H 3×CH2), 3.77 (s, 3H, OCH3), 3.26 (d, J = 6.8, 2H, CH2), 2.96-2.93 (t, 4H, 2×CH2), 2.662.50 (m, 36H, 12×CH2, 4×CH3), 1.93-1.78 (14H, 4×CH2, 2×CH3), 1.70 (s, 3H, CH3), 1.66 (s, 3H, CH3).

13

C NMR (101 MHz, MeOD) δ 183.15, 164.19, 160.50, 158.93, 156.59, 156.44,

145.42, 137.74, 131.89, 131.76, 125.01, 123.93, 112.67, 112.36, 104.51, 100.16, 90.60, 69.85, 69.40, 61.34, 59.17, 59.10, 55.94 (2×CH2), 54.69 (2×CH2), 53.79 (2×CH2), 53.77 (2×CH2), 53.70 (2×CH2), 53.67 (2×CH2), 44.64 (4×CH3), 28.29, 28.11, 27.04, 26.11, 26.04, 24.13, 24.07, 22.37, 18.42, 18.15. MS (ESI): calcd for C48H77N6O6 [M + H]+ 833.6, found 833.6. Di-tert-butyl

4,4'-(((1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9-oxo-9H-

xanthene – 3,6-diyl)bis(oxy))bis(butane-4,1-diyl))bis(piperazine-1-carboxylate) (5k) was prepared according to general procedure C from 1c (100 mg, 0.15 mmol) and 1-Bocpiperazine (546 mg). 5k was obtained as a light yellow oil (118 mg) in 90% yield. Purity: 99.7%. 1H NMR (400 MHz, CDCl3) δ 13.49 (s, 1H, OH), 6.71 (s, 1H, Ar-H), 6.28 (s, 1H, Ar-



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H), 5.24-5.21 (m, 2H, 2×CH), 4.12-4.06 (m, 6H, 3×CH2), 3.79 (s, 3H, OCH3), 3.47-3.42 (m, 8H, 4×CH2), 3.35 (d, J = 7.2, 2H, CH2), 2.44-2.39 (m, 12H, 6×CH2), 1.95-1.86 (m, 4H, 2×CH2), 1.84 (s, 3H, CH3), 1.79 (s, 3H, CH3), 1.76-1.71 (m, 4H, 2×CH2), 1.67 (s, 6H, 2×CH3), 1.45 (s, 18H, 6×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.12, 162.87, 160.03,

157.51, 155.42, 155.29, 154.91 (2×CO), 144.21, 137.41, 131.83, 131.49, 123.43, 122.67, 112.14, 111.64, 104.05, 98.89, 89.34, 79.78, 79.75, 68.75, 68.31, 60.98, 58.25 (2×CH2), 58.15 (2×CH2), 53.13 (6×CH2), 28.57 (6×CH2), 27.21, 27.03, 26.33, 26.04, 25.96, 23.43, 23.41, 21.61, 18.32, 17.98. MS (APCI): calcd for C50H75N4O10 [M + H]+ 891.5, found 891.6. 3,6-Bis(4-(1H-imidazol-1-yl)butoxy)-1-hydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1yl)-9H-xanthen-9-one (6a) was prepared according to general procedure D from 1c (100 mg, 0.15 mmol) and 1H-imidazole (100 mg, 1.47 mmol). 6a was obtained as a light yellow solid (70 mg) in 73% yield. Purity: 98.4%. Mp 74–76 °C. 1H NMR (400 MHz, CDCl3) δ 13.46 (s, 1H, OH), 7.66 (s, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.09 (s, 2H, Ar-H), 6.96 (s, 1H, Ar-H), 6.94 (s, 1H, Ar-H), 6.66 (s, 1H, Ar-H), 6.24 (s, 1H, Ar-H), 5.24-5.17 (m, 2H, 2×CH), 4.12-4.01 (m, 10H, 5×CH2), 3.76 (s, 3H, OCH3), 3.33 (d, J = 6.8, 2H, CH2), 2.09-2.00 (m, 4H, 2×CH2), 1.96-1.82 (m, 7H, 2×CH2, 1×CH3), 1.77 (s, 3H, 1×CH3), 1.67 (s, 3H, 1×CH3), 1.65 (s, 3H, 1×CH3).

13

C NMR (101 MHz, CDCl3) δ 182.06, 162.49, 160.05, 157.19, 155.33, 155.20,

144.15, 137.57, 137.10, 137.06, 131.92, 131.63, 129.06, 129.04, 123.29, 122.57, 118.94, 118.89, 104.13, 98.95, 89.32. 13C NMR (101 MHz, CDCl3) δ 182.06, 162.49, 160.05, 157.19, 155.33, 155.20, 144.15, 137.57, 137.10, 137.06, 131.92, 131.63, 129.06, 129.04, 123.29, 122.57, 118.94, 118.89, 112.32, 111.60, 104.13, 98.95, 89.32, 68.14, 67.57, 61.03, 47.01, 46.94, 28.07, 27.92, 26.33, 26.30, 26.17, 26.01, 25.90, 21.57, 18.30, 17.97. MS (ESI): calcd for C38H47N4O6 [M + H]+ 655.3, found 655.3. 1-Hydroxy-3,6-bis(4-(pyrrolidin-1-yl)butoxy)-9H-xanthen-9-one

(8b)

was

prepared

according to general procedure E from 7a (100 mg, 0.194 mmol) and pyrrolidine (4 mL). 8b


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was obtained as a light yellow oil (74 mg) in 77% yield. Purity: 98.2%. 1H NMR (400 MHz, CDCl3) 12.97 (s, 1H, OH), δ 8.11 (d, J = 8.8, 1H, Ar-H), 6.90 (dd, J = 2.4, 8.8, 1H, Ar-H), 6.80 (d, J = 2.4, 1H, Ar-H), 6.37 (d, J = 2, 1H, Ar-H), 6.31 (d, J = 2, 1H, Ar-H), 4.10-4.04 (m, 4H, 2×CH2), 2.55-2.50 (m, 12H, 6×CH2), 1.91-1.82 (m, 4H, 2×CH2), 1.81-1.76 (m, 8H, 4×CH2), 1.74-1.68 (m, 4H, 2×CH2). 13C NMR (101 MHz, CDCl3) δ 179.42, 165.07, 164.09, 162.81, 157.33, 157.07, 126.58, 113.53, 112.79, 102.82, 99.96, 96.66, 92.53, 67.85, 67.72, 55.33, 55.31, 53.50 (4×CH2), 26.42, 26.41, 24.70 (2×CH2), 22.77 (4×CH2). MS (ESI): calcd for C29H39N2O5 [M + H]+ 695.3, found 495.2. 3,6-Bis(4-(dimethylamino)butoxy)-1-hydroxy-2,8-diisopentyl-7-methoxy-9H-xanthen-9one (10b) was prepared according to general procedure F from 9a (100 mg, 0.15 mmol) and dimethyl amine (4 mL). 10b was obtained as a light yellow oil (62 mg) in 69% yield. Purity: 98.7%. 1H NMR (400 MHz, CDCl3) δ 13.65 (s, 1H, OH), 6.70 (s, 1H, Ar-H), 6.28 (s, 1H, ArH), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.83 (s, 3H, OCH3), 3.37-3.33 (m, 2H, CH2), 2.672.63 (m, 2H, CH2), 2.42-2.37 (m, 4H, 2 ×CH2), 2.28 (m, 12H, 4×CH3), 1.98-1.84 (m, 5H, 2×CH2, 1×CH), 1.77-1.69 (m, 4H, 2×CH2), 1.61-1.58 (m, 1H, CH), 1.47-1.37 (m, 4H, 2×CH2), 1.00 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.94 (s, 3H, CH3) 13C NMR (101 MHz, CDCl3) δ 181.33, 162.14, 159.34, 156.57, 154.69, 154.33, 138.73, 122.66, 112.14, 111.41, 103.15, 97.81, 88.37, 67.93, 67.40, 60.47, 58.58, 58.55, 44.67 (2×CH3), 44.63 (2×CH3), 39.65, 37.51, 28.19, 27.67, 26.36, 26.21, 24.45, 23.49, 23.43, 22.00 (2×CH3), 21.88 (2×CH3), 19.58. MS (ESI): calcd for C36H57N2O6 [M + H]+ 613.4, found 613.4. 1-Hydroxy-2,8-diisopentyl-7-methoxy-3,6-bis(4-(pyrrolidin-1-yl)butoxy)-9H-xanthen-9one (10c) was prepared according to general procedure F from 9a (100 mg, 0.15 mmol) and pyrrolidine (4 mL). 10c was obtained as a light yellow oil (78 mg) in 80% yield. Purity: 95.1%. 1H NMR (400 MHz, CDCl3) δ 13.65 (s, 1H, OH), 6.70 (s, 1H, Ar-H), 6.28 (s, 1H, ArH), 4.10 (t, 2H, CH2), 4.05 (t, 2H, CH2), 3.82 (s, 3H, CH3), 3.37-3.33 (m, 2H, CH2), 2.67-



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2.63 (m, 2H, CH2), 2.61-2.56 (m, 12H, 6×CH2), 1.99-1.94 (m, 2H, CH2), 1.92-1.86 (m, 2H, CH2), 1.84-1.72 (m, 13H, 6×CH2, 1×CH), 1.64-1.58 (m, 1H, CH), 1.47-1.37 (m, 4H, 2×CH2), 1.00 (s, 3H, CH3), 0.99 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.94 (s, 3H, CH3).

13

C NMR (101

MHz, CDCl3) δ 182.33, 163.14, 160.33, 157.56, 155.69, 155.32, 144.19, 139.70, 113.12, 112.40, 104.14, 98.81, 89.37, 68.97, 68.44, 61.48, 56.38, 56.35, 54.50 (2×CH2), 54.47 (2×CH2), 40.65, 38.50, 29.18, 28.66, 27.57, 27.41, 25.74, 25.72, 25.45, 23.77 (4×CH2), 23.00 (2×CH2), 22.88 (2×CH2), 20.58. MS (ESI): calcd for C40H61N2O6 [M + H]+ 665.5, found 665.4. 1,3,6-Tris(4-iodobutoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (11) was prepared according to general procedure G from α-mangostin (500 mg, 1.22 mmol) and 1,4-diiodobutane (2.41 mL, 18.3 mmol). 11 was obtained as a light yellow solid (723 mg) in 62% yield. Purity: 96.0%. Mp 100–102 °C. 1H NMR (400 MHz, CDCl3) δ 13.48 (s, 1H, OH), 6.69 (s, 1H, Ar–H), 6.27 (s, 1H, Ar–H), 5.28 – 5.23 (m, 2H, 2×CH), 4.19-4.01 (m, 8H, 4×CH2), 3.80 (s, 3H, OCH3), 3.37-3.33 (m, 2H, CH2), 3.32-3.22 (m, 6H, 3×CH2), 2.16-1.92 (m, 12H, 6×CH2), 1.85 (s, 3H, CH3), 1.80 (s, 3H, CH3),1.69 (s, 6H, 2×CH3). 13C NMR (101 MHz, CDCl3) δ181.89, 162.48, 159.80, 157.14, 155.14, 155.01, 143.97, 137.20, 131.64, 131.38, 123.23, 122.45, 112.00, 111.44, 103.88, 98.71, 89.15, 67.54 (2×CH2), 67.05, 60.88, 30.03 (2×CH2), 29.99 (2×CH2), 29.81 (2×CH2), 26.14, 25.87, 25.80, 21.41, 18.16, 17.87, 6.03 (2×CH2), 5.94. MS (APCI): calcd for C36H48I3O6 [M + H]+ 957.1, found 957.0. 1,3,6-Tris(4-(diethylamino)butoxy)-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9Hxanthen-9-one (12) was prepared according to general procedure G from 11 (100 mg, 0.11 mmol) and diethylamine (4 mL). 12 was obtained as a light yellow oil (28 mg) in 34% yield. Purity: 96.5%. 1H NMR (400 MHz, CDCl3) δ 6.99 (s, 1H, Ar–H), 6.87 (s, 1H, Ar–H), 5.26 (t, 1H, CH), 5.16 (t, 1H, CH), 4.28-4.17 (m, 4H, 2×CH2), 4.13-4.07 (m, 2H, CH2), 3.99-3.93 (m, 2H, CH2), 3.80 (s, 3H, OCH3), 3.46-3.38 (m, 4H, 2×CH2), 3.30-3.18 (m, 16H, 8×CH2), 2.15-


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1.92 (m, 12H, 6×CH2), 1.84 (s, 3H, CH3), 1.83 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.38-1.27 (m, 18H, 9×CH3).

13

C NMR (101 MHz, MeOD) δ 178.51, 163.38, 158.21,

157.99, 155.95, 145.59, 137.74, 132.50, 132.03, 125.34, 124.21, 122.17, 115.23, 111.59, 100.16, 96.86, 75.19, 69.44, 69.21, 61.46, 53.67, 52.57, 52.52, 48.49 (2×CH2), 48.19 (2×CH2), 48.16 (2×CH2), 28.71, 27.35, 27.18, 26.96, 26.08, 25.92, 23.57, 22.61, 22.12, 21.89, 18.41, 18.36, 9.14 (2×CH3), 9.07 (4×CH3). MS (ESI): calcd for C48H78N3O6 [M + H]+ 792.6, found 792.4. Bacterial strains and growth media. The bacterial strains used in these studies were clinical isolate MRSA DM21455 (isolated from eye), S. aureus ATCC29213, clinical isolate S. aureus DM4001 (isolated from eye) and Bacillus cereus ATCC11778. Inoculum suspensions were prepared from isolated colonies using the direct colony suspension method described by the Clinical and Laboratory Institute (CLSI). Colonies were selected from an 18- to 20-hour Tryptic Soy Agar (TSA) plate. Determination of antimicrobial activity. Susceptibility testing of all compounds was performed in Mueller Hinton Broth (MHB) using the broth macro-dilution in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines and our previous report.20 Briefly, compounds were dissolved in DMF to prepare 1,000 µg/mL stock solutions. Cationadjusted MHB (CA-MHB) was used to prepare serial twofold dilutions of the stock solutions in test tubes. Aliquots of a suspension containing bacteria at a concentration of 106 Colony Forming Units (CFU)/mL in MHB were added to the diluted solutions containing compounds. After inoculation, each tube contained approximately 5× 105 CFU/mL. The dilutions were tested in duplicate. Bacteria were incubated with the compounds for 24 h at 35 °C prior to reading.



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Determination of hemolytic activity. The hemolysis assay was performed as described previously.20 Briefly, fresh rabbit red blood cells (RBCs) were isolated. All procedures to isolate blood from New Zealand white rabbits were approved by IACUC Singhealth and performed according to the standards of the Association for Research in Vision and Ophthalmology. RBCs were isolated from serum by centrifugation at 3,000 rpm for 10 min and then washed twice with sterile PBS. Xanthone analogs dissolved at the desired concentrations in DMF were mixed with the RBCs (final v/v of RBCs = 4%). The mixtures were then incubated at 37 °C for 1 h in a 96-well plate (SPL life science). After the incubation period, the mixtures were carefully transferred to 1.5-mL Eppendorf tubes and centrifuged at 3,000 rpm for 3 min. Then, the supernatant (100 µL) was transferred to a clean 96-well plate, and hemoglobin release was determined by measuring the absorbance at 576 nm in a TECAN Infinite 200 microplate reader. Triton X-100 (2%) was used as a positive control. DMF (1%) were used as negative controls. The following formula was used to calculate the % hemolysis:

% Hemolysis =

− 100 −

SYTOX green uptake. SYTOX Green is a high-affinity nucleic acid stain that can easily penetrate cells with compromised plasma membranes but cannot cross the intact membranes of live cells. The protocol was modified from the method of Rathinakumar et al.16b Briefly, a bacterial culture (clinical isolate S. aureus DM4001) was harvested at the exponential phase. The bacteria were then suspended and washed twice with sterile 20 mM PBS buffer (pH 7) until an optical density of 0.09 at 620 nm was obtained. Then, 3 µM SYTOX Green was added to the bacterial suspension. The mixture was incubated in the dark, and the fluorescence signal was monitored in a stirring cuvette at an excitation wavelength of 504 nm and emission wavelength of 523 nm until the fluorescence signal was stabilized. Then, 10 µM 48 ACS Paragon Plus Environment

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xanthone analog was added, and the increase in SYTOX green fluorescence intensity was measured. Melittin at 10 µg/mL was used as a positive control. Experiments were repeated at least twice, and the results were reproducible. Data from one experiment are presented. Molecular hydrophobicity analysis. Molecular hydrophobicity was characterized in terms of retention time and ACN% by HPLC (Waters 2695 separation module, Waters Delta-Pak CA 300 Å column). The experiments were conducted under the same conditions for all analogs. The samples were injected at 10 µg/mL with an injection volume of 20 µL and a flow rate of 1 mL/min. The gradient profile used was 5-95% in 15 min. Vesicle leakage from calcein-loaded LUV. All phospholipids used in this assay were purchased from Avanti Polar Lipids, Inc. (Alabaster), and used without further purification. The phospholipids used in this study were 1,2-di-(9Z-octadecenoyl)-sn-glycero-3phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DOPG) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC). Calceinloaded large unilamellar vesicles (LUVs) were prepared using a film hydration method as described previously.8a Briefly, the phospholipids were dissolved in methanol-chloroform (1:2 by volume) and transferred to a test tube. Then, the phospholipid solution was dried gently using a constant stream of nitrogen gas and placed under vacuum for at least 2 h. Calcein solution (80 mM calcein, 50 mM HEPES, 100 mM NaCl and 0.3 mM EDTA; pH 7.4) was added to the dried phospholipid film to a final phospholipid concentration of 30 mM. The solution was then frozen in liquid nitrogen and warmed in a water bath for 7 cycles. A mini-extruder (Avanti Polar Lipid Inc.) was used to prepare 100-nm homogeneous LUVs. The extrusion was repeated for 15 cycles using a polycarbonate membrane (Whatman) with a pore size of 100 nm. The vesicles were then passed through a Sephadex G-50 gel filtration column to remove excess free calcein. The concentration of the calcein-encapsulated liposomes was determined using a total phosphorus assay. An aliquot of the calcein49 ACS Paragon Plus Environment


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encapsulated LUVs was transferred to a stirred cuvette. Desired concentrations of xanthone analogs were prepared in DMF and added to obtain lipid to compound ratios of 2, 4, 8, 16 and 32. The final concentration of phospholipids was 50 µM. The percentage of DMF in any experiment was less than 0.2%. Control experiments with 0.2% DMF confirmed that the LUVs were not lysed in the presence of DMF. To determine the intensity at 100% lysis, 0.1% Triton X-100 was added. The fluorescence emission intensity was monitored using a TECAN infinite M200Pro micro-plate reader at an excitation wavelength of 490 nm and an emission wavelength of 520 nm for 30 min. The percentage of leakage (%L) was calculated using the following formula: %L = [(It – Io)/ (I∞-Io)]×100%, where Io and It are the intensities before and after the addition of xanthone analogs and I∞ is the intensity after the addition of 0.1% Triton X-100. Quantification of the membrane-damaging effect of xanthone analogs. Bacterial membrane damage was assessed using Live/Dead® BacLightTM bacterial viability kits (Molecular Probes, Invitrogen) with minor modifications to the manufacturer’s protocol. In this assay, green-fluorescent SYTO-9 stain and red-fluorescent propidium iodide stain were employed to distinguish intact and damaged bacterial cell membranes. SYTO-9 stain labels cells with both intact and damaged membranes. By contrast, propidium iodide stain only penetrates and labels bacteria with damaged membranes. Thus, bacteria with intact membranes fluoresce green, whereas bacteria with damaged membranes fluoresce red. Briefly, clinical isolate S. aureus DM4001 was grown overnight and harvested at the exponential phase. The culture was washed twice with 0.9% saline (live culture). A portion of the culture was re-suspended in 70% 2-propanol to prepare a “dead” culture containing cells with fully permeabilized membranes. Both the live and dead cultures were incubated at room temperature for 1 h. Both cultures were then washed twice and re-suspended in 0.9% saline until an OD670 of 0.3 was obtained. Bacterial suspensions from the live culture were 50 ACS Paragon Plus Environment

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incubated with the desired concentrations of xanthone analogs at room temperature for 10 min. At the end of the incubation period, the suspension was centrifuged at 4000 g for 5 min. The cells were re-suspended in the same volume of 0.9% saline, and 100 µL of the bacterial suspension was added to a 96-well plate. An equal volume of BacLight reagent (10 µM of SYTO 9 stain and 60 µM of propidium iodide) was added to each well. Then, the plate was incubated in the dark for 15 min. At an excitation wavelength of 485 nm, the emission intensity from 500 nm – 700 nm was recorded using a TECAN infinite M200Pro microplate reader. The green to red ratio (G/R) was determined using the following formula: ∑//012 34 F*+,,,+. G ratio = /052 34 R ∑ 602 34 F*+,,,+. /672 34

and the % of membrane damage was quantified using a G/R ratio standard curve generated using bacterial mixtures of 0, 10%, 50%, 90% and 100% dead culture. Extracellular ATP bioluminescence assay. An ATP determination kit (Molecular Probes, Invitrogen) was used to measure extracellular ATP levels released by the bacteria. In this assay, the production of light by luciferase is dependent on ATP released from bacteria and can be detected by a luminometer. The measurement was performed as described by the manufacturer’s instructions, with the following modification. Briefly, the clinical isolate S. aureus DM4001 was grown to exponential phase and washed twice with 20 mM PBS buffer (pH 7). The bacterial culture was then re-suspended in the same buffer until an OD620 of 0.3 was obtained. Following the addition of each xanthone analog at the desired concentration, the bacteria were incubated at 37 °C for 10 min. After the incubation period, the treated cells were centrifuged at 4000 r.p.m. for 5 min. An aliquot (100 µL) of the supernatant was transferred to a sterile white immuno-96-well plate (SPL Life Sciences). Then, 100 µL of enzyme mixture (1× reaction buffer, 1 mM dithiothreitol, 0.5 mM D-luciferin and 1.25 51 ACS Paragon Plus Environment


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µg/mL firefly luciferase) was added. The concentration of extracellular ATP was measured using a TECAN Infinite M200Pro microplate luminometer for 30 cycles. Aggregation study using molecular dynamic (MD) simulations. To investigate the aggregation behavior of xanthone analogs with different chain lengths, the association free energies of xanthone analogs with spacer lengths of n = 2, 3, 6, 8 and 12 were calculated using atomistic MD simulations. The xanthone analogs were modeled using the Gromos53a6 force field, and the SPC model was used to model water molecules. Two monomers were initially solvated in a cubic box, and 500 steps of energy minimization and 200 ps equilibrium in NPT ensemble were performed. The free energies were calculated using metadynamics with the distance between two monomers as the collective variable. During the metadynamics simulations, Gaussian potentials with a width of 0.3 nm and a height of 0.5 kJ/mol were added to the Hamiltonian until the system underwent a random walk along the distance between the two monomers. In each simulation, van der Waals interaction and shortrange electrostatic interactions cut-offs of 1.4 nm and 0.9 nm, respectively, were used, while long-range electrostatic interactions were calculated using PME. Time-kill kinetics. In this study, a time-kill kinetic assay was used to determine the rate of bacterial killing at specific drug concentrations. The bacterial strains (S. aureus DM4001 and MRSA DM21455) were prepared from isolated colonies on an 18-22 h TSA plate and suspended in 0.31 mM phosphate buffer at pH 7.2 until a bacterial suspension of 105 to 106 CFU/mL was obtained. The inoculum was then treated with 3a and vancomycin at 1×, 2× and 4× MIC. The mixtures were incubated at 35 ºC, and culture aliquots were collected at 10 min, 30 min, 1 h, 2 h and 5 h to measure the viable plate counts. The aliquots were serially diluted ten-fold using phosphate buffer, and 100 µL of each dilution was plated with MuellerHinton Agar (MHA) using the surface-spread plate method. The plates were then incubated at


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35 ºC for 48 to 72 h. The number of viable bacteria was determined by counting the colonies that grew on the plates. The detection limit of the reliable viable count method was 100 CFU/mL. Bactericidal activity was defined as a 3-log reduction in the viable count in a sample treated with 3a compared to an untreated control. Luminescent Cell Viability Assay. Human corneal fibroblasts were plated at a density of 10,000 cells per well in a 96-well opaque white plate (SPL Life Sciences Inc., Korea). Then, the desired concentrations of 3a were added. The final volume in each well was 100 µL. The mixtures were incubated for 4 h. After incubation, the plate was equilibrated to 22 ºC for 30 min. CellTiter-Glo reagent (Promega Inc., USA) was added to each well, and the cell viability assay was performed according to the manufacturer’s instructions. Luminescence was assessed using a TECAN Infinite M200 Pro micro-plate reader. Cells treated with 1% Triton X-100 were used as the positive control to represent the minimal viability. DMF at 0.5% was used as the negative control. Cell viability was determined using the formula below: % Viable Cells = 8

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